U.S. patent number 8,435,513 [Application Number 13/003,013] was granted by the patent office on 2013-05-07 for notch1 receptor antibodies and methods of treatment.
This patent grant is currently assigned to OncoMed Pharmaceuticals, Inc.. The grantee listed for this patent is Fumiko Takada Axelrod, Maureen Fitch Bruhns, Austin L. Gurney, Timothy Hoey. Invention is credited to Fumiko Takada Axelrod, Maureen Fitch Bruhns, Austin L. Gurney, Timothy Hoey.
United States Patent |
8,435,513 |
Gurney , et al. |
May 7, 2013 |
NOTCH1 receptor antibodies and methods of treatment
Abstract
The present invention relates to compositions and methods for
characterizing, diagnosing, and treating cancer. In particular the
invention provides the means and methods for the diagnosis,
characterization, prognosis and treatment of cancer and
specifically targeting cancer stem cells. The present invention
provides an antibody that specifically binds to a non-ligand
binding membrane proximal region of the extracellular domain of a
human Notch receptor and inhibits tumor growth. The present
invention further provides a method of treating cancer, the method
comprising administering a therapeutically effective amount of an
antibody that specifically binds to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch
receptor protein and inhibits tumor growth.
Inventors: |
Gurney; Austin L. (San
Francisco, CA), Hoey; Timothy (Hillsborough, CA), Bruhns;
Maureen Fitch (San Mateo, CA), Axelrod; Fumiko Takada
(Palo Alto, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Gurney; Austin L.
Hoey; Timothy
Bruhns; Maureen Fitch
Axelrod; Fumiko Takada |
San Francisco
Hillsborough
San Mateo
Palo Alto |
CA
CA
CA
CA |
US
US
US
US |
|
|
Assignee: |
OncoMed Pharmaceuticals, Inc.
(Redwood City, CA)
|
Family
ID: |
41507637 |
Appl.
No.: |
13/003,013 |
Filed: |
July 8, 2009 |
PCT
Filed: |
July 08, 2009 |
PCT No.: |
PCT/US2009/003995 |
371(c)(1),(2),(4) Date: |
April 07, 2011 |
PCT
Pub. No.: |
WO2010/005567 |
PCT
Pub. Date: |
January 14, 2010 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20110311552 A1 |
Dec 22, 2011 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
61079095 |
Jul 8, 2008 |
|
|
|
|
61112699 |
Nov 7, 2008 |
|
|
|
|
61112701 |
Nov 7, 2008 |
|
|
|
|
Current U.S.
Class: |
424/130.1;
435/325; 435/334; 530/388.22; 514/19.2; 530/388.1; 514/19.4;
424/143.1; 530/387.1; 435/328; 424/141.1; 424/135.1; 424/133.1;
530/389.1; 435/326; 530/387.3; 424/136.1; 536/23.1; 514/19.3;
536/23.5; 514/19.5 |
Current CPC
Class: |
C07K
16/462 (20130101); A61N 5/10 (20130101); C07K
16/30 (20130101); C07K 16/28 (20130101); C07K
16/464 (20130101); C12N 5/0693 (20130101); A61P
35/00 (20180101); A61K 31/337 (20130101); A61P
43/00 (20180101); A61K 39/3955 (20130101); C07K
16/3046 (20130101); A61K 45/06 (20130101); A61P
9/00 (20180101); A61P 35/04 (20180101); A61K
39/39558 (20130101); C07K 16/22 (20130101); C07K
2317/76 (20130101); C07K 2317/24 (20130101); C12N
2501/998 (20130101); C07K 2317/34 (20130101); C07K
2317/734 (20130101); C12N 15/63 (20130101); C07K
2317/92 (20130101); C07K 2317/73 (20130101); C07K
14/71 (20130101); C12N 5/12 (20130101); C07K
2317/21 (20130101); C07K 14/435 (20130101); C07K
2317/732 (20130101); C07H 21/04 (20130101); C12N
15/79 (20130101); C07K 2317/565 (20130101); A61K
2039/505 (20130101); C07K 2317/56 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); C12N 5/00 (20060101); C12N
5/10 (20060101); C07H 21/04 (20060101); C07K
16/28 (20060101); C07K 16/18 (20060101); C07K
16/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
44 25 115 |
|
Jan 1996 |
|
DE |
|
0 662 827 |
|
Jul 1995 |
|
EP |
|
2002-526109 |
|
Aug 2002 |
|
JP |
|
WO 94/07474 |
|
Apr 1994 |
|
WO |
|
WO 97/37004 |
|
Oct 1997 |
|
WO |
|
WO 97/45143 |
|
Dec 1997 |
|
WO |
|
WO 98/57621 |
|
Dec 1998 |
|
WO |
|
WO 00/20576 |
|
Apr 2000 |
|
WO |
|
WO 00/52143 |
|
Sep 2000 |
|
WO |
|
WO 02/00576 |
|
Jan 2002 |
|
WO |
|
WO 02/12447 |
|
Feb 2002 |
|
WO |
|
WO 02/18544 |
|
Mar 2002 |
|
WO |
|
WO 03/042246 |
|
May 2003 |
|
WO |
|
WO 03/050502 |
|
Jun 2003 |
|
WO |
|
WO 03/062273 |
|
Jul 2003 |
|
WO |
|
WO 2004/001004 |
|
Dec 2003 |
|
WO |
|
WO 2004/052389 |
|
Jun 2004 |
|
WO |
|
WO 2004/091383 |
|
Oct 2004 |
|
WO |
|
WO 2004/094475 |
|
Nov 2004 |
|
WO |
|
WO 2005/026334 |
|
Mar 2005 |
|
WO |
|
WO 2005/054434 |
|
Jun 2005 |
|
WO |
|
WO 2005/074633 |
|
Aug 2005 |
|
WO |
|
WO 2006/110581 |
|
Oct 2006 |
|
WO |
|
WO 2007/145840 |
|
Dec 2007 |
|
WO |
|
WO 2008/051797 |
|
May 2008 |
|
WO |
|
WO 2008/057144 |
|
May 2008 |
|
WO |
|
WO 2008/076960 |
|
Jun 2008 |
|
WO |
|
WO 2008/091641 |
|
Jul 2008 |
|
WO |
|
WO 2008/108910 |
|
Sep 2008 |
|
WO |
|
WO 2008/136848 |
|
Nov 2008 |
|
WO |
|
WO-2008150525 |
|
Dec 2008 |
|
WO |
|
WO 2009/025867 |
|
Feb 2009 |
|
WO |
|
WO 2009/035522 |
|
Mar 2009 |
|
WO |
|
WO 2010/005567 |
|
Jan 2010 |
|
WO |
|
Other References
Brummell et al. (Biochemistry 32:1180-1187 (1993)). cited by
examiner .
Kobayashi et al. (Protein Engineering 12:879-844 (1999)). cited by
examiner .
Burks et al. (PNAS 94:412-417 (1997)). cited by examiner .
Jang et al. (Molec. Immunol. 35:1207-1217 (1998)). cited by
examiner .
Brorson et al. (J. Immunol. 163:6694-6701 (1999)). cited by
examiner .
Coleman (Research in Immunol. 145:33-36 (1994)). cited by examiner
.
Rudikoff et al, 1982 (Proc Natl Acad Sci USA. vol. 79: 1979-1983).
cited by examiner .
MacCallum et al (1996. J Mol Biol. 262: 732-745). cited by examiner
.
de Pascalis et al (2002. The Journal of Immunology. 169:
3076-3084). cited by examiner .
Casset et al (2003. Biochemical and Biophysical Research
Communications. 307: 198-205). cited by examiner .
Vajdos et al (2002. J Mol Biol. 320: 415-428). cited by examiner
.
Holm et al (2007. Mol Immunology. 44: 1075-1084). cited by examiner
.
Chen et al (1999. J Mol Biol. 293: 865-881). cited by examiner
.
Wu et al (1999. J Mol Biol. 294: 151-162). cited by examiner .
Paul, W.E. Fundamental Immunology, 3rd Edition, Raven Press, New
York, Chapt. 8, pp. 292-295 (1993). cited by examiner .
Al-Hajj et al., "Prospective Identification of Tumorigenic Breast
Cancer Cells," Cell Biology 100:3983-3988 (2003), Proceedings of
the National Academy of Science, 700 11th Street, NW Suite 450
Washington, DC 20001. cited by applicant .
Arias et al., "Csl-Independent Notch Signalling: A Checkpoint in
Ceu Fate Decisions During Development?," Current Opinion In
Genetics & Development, 12:524-533 (2002), Elsevier Science
Ltd, The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1 GB,
UK. cited by applicant .
Artavanis-Tsakonas et al., "Notch Signaling: Cell Fate Control and
Signal Integration in Development," Science 284:770-776 (1999),
American Association for the Advancement of Science, 1200 New York
Avenue, NW, Washington, DC 20005. cited by applicant .
Brennan and Brown, "Is There a Role for Notch Signalling in Human
Breast Cancer?," Breast Cancer Research, 5:69-75 (2003), BioMed
Central Ltd, London WC1X 8HL, United Kingdom. cited by applicant
.
Brennan et al., "Repression by Notch is Required Before Wingless
Signalling During Muscle Progenitor Cell Development in
Drosophila," Current Biology, 9:707-710 (1991), Current Biology
Publications, 34-42 Cleveland Street, London W1 P GLE, UK. cited by
applicant .
Cole et al., "The Ebv-Hybridoma Technique and its Application to
Human Lung Cancer," Monoclonal Antibodies and Cancer Therapy, 77-96
(1985), Alan R. Liss, Inc, 41 East 11th Street, New York, NY 10003.
cited by applicant .
Del Amo et al., "Cloning, Analysis, and Chromosomal Localization of
Notch-1, a Mouse Homolog of Drospohila Notch," Genomics, 15:259-264
(1993), Academic Press, Inc. cited by applicant .
Domenga et al., "Notch3 is required for arterial identity and
maturation of vascular smooth muscle cells," Genes &
Development, 18:2730-2735 (2004), Cold Spring Harbor Laboratory
Press. cited by applicant .
Duncan et al., "Integration of Notch and Wnt Signaling in
Hematopoietic Stem Cell Maintenance," Nature Immunology 6:314-322
(2005), Nature Publishing Group, 345 Park Avenue South, New York,
NY 10010-1707. cited by applicant .
Ellisen et al., "Tan-1, The Human Homolog of the Drosophila Notch
Gene, Is Broken by Chromosomal Translocations in T Lymphoblastic
Neoplasms," Cell, 66:649-661 (1991), Cell Press, 50 Church Street,
Cambridge, Massachusetts 02138. cited by applicant .
Gale et al., "Haploinsufficiency of Delta-Like 4 Ligand Results in
Embryonic Lethality Due to Major Defects in Arterial and Vascular
Development," PNAS, 101:15949-15954 (2004), National Academy of
Science, 700 11th Street, NW Suite 450 Washington, DC 20001. cited
by applicant .
Gallahan et al., "Expression of a Truncated Int3 Gene in Developing
Secretory Mammary Epithelium Specifically Retards Lobular
Differentiation Resulting in Tumorigenesis," Cancer Research,
56:1775-1785 (1996), American Association for Cancer Research, Inc,
Public Ledger Bldg., Suite 816, 150 South Independence Mall West,
Philadelphia, PA 19106-3483. cited by applicant .
Gridley, T., "Notch signaling and inherited disease syndromes,"
Human Molecular Genetics 12:R9-R13 (2003), Oxford University Press.
cited by applicant .
Gridley, T., "Notch signaling during vascular development," PNAS,
98:5377-5378 (2001), National Academy of Sciences, Washington, DC
20001. cited by applicant .
Gridley, T., "Vessel guidance," Nature, 445:722-723 (2007) Nature
Publishing Group, New York, NY 10013-1917, USA. cited by applicant
.
Gridley, T., "Notch Signaling in Vertebrate Development and
Disease," Mol. Cell. Neurosci, 9:103-108, (1997), Academic Press,
6377 Sea Harbor Drive, Orlando, FL 32887-4900. cited by applicant
.
Hadland et al., "A requirement for Notch1 distinguishes 2 phases of
definitive hematopoiesis during development," Blood, 104:3097-3105
(2004), The American Society of Hematology. cited by applicant
.
Hainaud et al., "The Role of the Vascular Endothelial Growth
Factor-Delta-like 4 Ligand/Notch4-Ephrin B2 Cascade in Tumor Vessel
Remodeling and Endothelial Cell Functions," Cancer Res,
66:8501-8510, (2006), American Association for Cancer Research.
cited by applicant .
Hallahan et al., "The SmoA1 Mouse Model Reveals that Notch
Signaling is Critical for the Growth and Survival of Sonic
Hedgehog-Induced Medulloblastomas," Cancer Research, 64:7794-7800
(2004), American Association for Cancer Research, Philadelphia, PA
19106-4404. cited by applicant .
Hitoshi et al., "Notch Pathway Molecules are Essential for Tile
Maintenance, but not the Generation of Mammalian Neural Stem
Cells," Genes & Development, 16:846-858 (2002), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor Laboratory Press 500
Sunnyside Boulevard Woodbury, New York 11797. cited by applicant
.
Iso et al., "Notch Signaling in Vascular Development," Arterioscler
Thrombosis and Vascular Biology, 23:543-553 (2003), Lippincott
Williams & Wilkins , Philadelphia, PA 19106. cited by applicant
.
Jhappan et al., "Expression of an Activated Notch-Related Int-3
Transgene Interferes with Cell Differentiation and Induces
Neoplastic Transformation in Mammary and Salivary Glands," Genes
& Development, 6:345-355 (1992), Cold Spring Harbor Laboratory
Press, Box 100, 1 Bungtown Road, Cold Spring Harbor, New York
11724-2203. cited by applicant .
Joutel and Tournier-Lasserve., "Notch Signalling Pathway and Human
Diseases," Seminars in Cell & Departmental Biology, 9:619-625
(1998), Academic Press, Orlando, FL 32887. cited by applicant .
Joutel et al., "Notch3 Mutations in Cadasil, A Herediraty Adult
Onset Consition Causing Stroke and Dementia," Nature, 383:707-710
(1996), Macmillan Magazines Ltd., 4 Little Essex Street, London
WC2R 3LF. cited by applicant .
Karanu et al., "The Notch Ligand Jagged-L Represents a Novel Growth
Factor of Human Hematopoietic Stem Cells," J. Exp. Med, 192:
1365-1372 (Nov. 6, 2000), The Rockefeller University Press, 1114
First Avenue, New York, 10021. cited by applicant .
Kidd et al., "Sequence of the Notch Locus of Drosophila
Melanogaster: Relationship of the Encoded Protein to Mammalian
Clotting and Growth Factors," Molecular and Cellular Biology,
6:3094-3108 (1986), American Society for Microbiology, 1913 I St.,
NW, Washington, DC 20006. cited by applicant .
Kopper and Hajdu "Tumor Stem Cells," Pathology Oncology Research,
10:69-73 (2004), Aranyi Lajor Foundation, Budapest. cited by
applicant .
Krebs et al., "Notch Signaling is Essential for Vascular
Morphogenesis in Mice," Genes & Development, 14:1343-1352
(2000), Cold Spring Harbor Laboratory Press, Box 100, 1 Bungtown
Road, Cold Spring Harbor, New York 11724-2203. cited by applicant
.
Kuukasjarvi et al., "Genetic Heterogeneity and Clonal Evolution
Underlying Development of Asynchronous Metastasis in Human Breast
Cancer," Cancer Research, 57:1597-1604 (Apr. 15, 1997), American
Association for Cancer Research, Inc., P.O. Box 3000, Denville, NJ
7834. cited by applicant .
Lapidot et al., "A Cell Initiating Human Acute Myeloid Leukaemia
after Transplantation into SCID Mice," Nature, 367:645-648 (1994),
Macmillan Magazines Ltd., 4 Little Essex Street, London WC2R 3LF.
cited by applicant .
Lawrence et al., "Notch Signaling Targets the Wingless
Responsiveness of a Ubx Visceral Mesoderm Enhancer in Drosophila,"
Current Biology, 11:375-385 (2001), Cell Press, 1100 Massachusetts
Avenue, Cambridge, MA 02138. cited by applicant .
Leethanakul et al., "Distinct Pattern of Expression of
Differentiation and Growth-Related Genes in Squamous Cell
Carcinomas of the Head and Neck Revealed by the Use of Laser
Capture Microdissection and Cdna Arrays," Oncogene, 19:3220-3224
(2000), Nature Publishing Group, Houndmills, Basingstoke, Hampshire
RG21 6XS, UK. cited by applicant .
Leong and Karsan, "Recent insights into the role of Notch signaling
in tumorigenesis," Blood, 107:2223-2233 (2006), American Society of
Hematology. cited by applicant .
Leong et al., "Activated Notch4 Inhibits Angiogenesis: Role of
.beta.1-Integrin Activartion," Mol. Cell. Biol., 22:2830-2841,
(2002) American Society for Microbiology. cited by applicant .
McCright et al., "Defects In Development of the Kidney, Heart and
Eye Vasculature in Mice Homozygous for a Hypomorphic Notch2
Mutation," Development, 128:491-501 (2001), The Company of
Biologists Limited, Bidder Building, 140 Cowley Road, Cambridge CB4
ODL, UK. cited by applicant .
Mohr, "Character Caused By Mutation of an Entire Region of a
Chromosome in Drosophila," Genetics, 4:275-292 (1919), The Genetics
Society of America, GENETICS Mellon Institute, Box I 4400 Fifth
Avenue Pittsburgh, Pennsylvania 15213-2683. cited by applicant
.
Parr et al., "The Possible Correlation of Notch-1 and Notch-2 with
Clinical Outcome and Tumor Clinicpathological Parameters in Human
Breast Cancer," International Journal of Molecular Medicine,
14:779-786 (2004), Springer Verlag, Tiergartenstasse 17, 69121
Heidelberg, Germany. cited by applicant .
Pear and Aster, "T Cell Acute Lymphoblastic Leukemia/Lymphoma: A
Human Cancer Commonly Associated with Aberrant Notch1 Signaling,"
Current Opinion in Hematology, 11:426-433 (2004), Lippincott
Williams & Wilkins, Philadelphia, PA 19106. cited by applicant
.
Politi et al., "Notch in Mammary Gland Development and Breast
Cancer," Seminars in Cancer Biology, 14:341-347 (2004), Academic
Press, 6277 Sea Harbor Drive, Orlando, FL, 32887-4900. cited by
applicant .
Purow et al., "Expression of Notch-1 and Its Ligands, Delta-Like-1
and Jagged-1, is Critical for Glioma Cell Survival and
Proliferation," Clinical Research, 65:2354-2363 (2005), American
Association for Cancer Research, Philadelphia, PA 19106-4404. cited
by applicant .
Rae et al., "Novel Association of a Diverse Range of Genes with
Renal Cell Carcinoma as Identified by Differential Display," Inter.
J. Cancer, 88:726-732 (2000), John Wiley & Sons, Inc, 350 Main
Street, Malden MA 02148, USA. cited by applicant .
Rebay et al., "Specific Egf Repeats of Notch Mediate Interactions
with Delta and Serrate: Implications for Notch as a Multifunctional
Receptor," Cell, 67:687-699 (1991), Cell Press, 50 Church Street,
Cambridge, Massachusetts 02138. cited by applicant .
Reya et al., "Stem Cells, Cancer and Cancer Stem Cells ,"Nature,
414:105-111 (2001), Nature Publishing Group, New York, NY
10013-1917, USA. cited by applicant .
Robey et al., "An Activated Form of Notch Influences the Choice
Between Cd4 and Cd8 T Cell Lineages," Cell, 87:483-492 (1996), Cell
Press, 50 Church Street, Cambridge, Massachusetts 02138. cited by
applicant .
Smith et al., "Constitutive Expression of a Truncated Int3 Gene in
Mouse Mammary Epithelium Impairs Differentiation and Functional
Development," Cell Growth & Differentiation, 6: 563-577 (1995),
American Association for Cancer Research, Philadelphia, PA
19106-4404. cited by applicant .
Soriano et al., "Expression of an Activated Notch4(Int-3)
Oncoprotein Disrupts Morphogenesis and Induces an Invasive
Phenotype in Mammary Epithelial Cells in Vitro," Intl. J. Cancer,
86: 652-659 (2000), John Wiley & Sons Inc, 350 Main Street,
Malden MA 02148, USA. cited by applicant .
Suzuki et al., "Imbalanced Expression of Tan-L and Human Notch4 in
Endometrial Cancers," International Journal of Oncology, 17:
1131-1139 (2000), Spandidos--publications, Athens 116 10, Greece.
cited by applicant .
Swiatek et al., "Notch1 is essential for postimplantation
development in mice," Genes & Development, 8:707-719, (1994)
Cold Spring Harbor Laboratory. cited by applicant .
Takeshita et al., "Crictical Role of Endothelial Notch1 Signaling
in Postnatal Angiogenesis," Cir. Res. 100:70-78 (2007), American
Heart Association, Inc. cited by applicant .
Tavares et al., "Inhibition of Vascular Endothelium by the
Notch-Ligand Delta-4 Unveils a Novel Therapeutic Target," Vascular
Wall Biology, Poster Board #--Session: 115-II, Abstract# 1944, pp.
531a, (2003), American Society of Hematology, San Diego,
California. cited by applicant .
Uyttendaele et al., "Notch4 and Wnt-L Proteins Function to Regulate
Branching Morphogenesis of Malnmary Epithelial Cells in an Opposing
Fashion," Developmental Biology, 196:204-217 (1998), Academic
Press, Orlando, FL 32887.cndot.4900. cited by applicant .
Van Es and Clevers, "Notch and Wnt Inhibitors as Potential New
Drugs for Intestinal Neoplastic Disease," Trends in Molecular
Medicine, 11: 496-502 (2005), Elsevier, London, UK WC1X 8RR. cited
by applicant .
Van Limpt et al., "Sage Analysis of Neuroblastoma Reveals a High
Expression of The Human Homologue of the Drosophila Delta Gene,"
Medical and Pediatric Oncology, 35:554-558 (2000), Wiley-Liss, Inc,
605 Third Avenue, New York, NY 10158-0012. cited by applicant .
Varnum-Finney et al., "Pluripotent, Cytokine-dependent,
Hematopoietic Stem Cells are Immortalized by Constitutive Notch1
Signaling," Nature Medicine, 6:1278-1281 (2000), Nature Publishing
Group, New York, NY 10013-1917, USA. cited by applicant .
Weijzen et al., "Activation of Notch-L Signaling Maintains the
Neoplastic Phenotype in Human Ras-Transformed Cells," Nature
Medicine, 8 :979-986 (2002), Nature Publishing Group, New York, NY
10013-1917, USA. cited by applicant .
Wharton et al., "Nucleotide Sequence from the Neurogenic Locus
Notch Implies a Gene Product that Shares Homology with Proteins
Containing Egf-Like Repeats," Cell, 43:567-581 (1985) , Cell Press,
50 Church Street, Cambridge, Massachusetts 02138. cited by
applicant .
Xu et al., "Regions of Drosophila Notch that Contribute to Ligand
Binding and the Modulatory Influence of Fringe," The Journal of
Biological Chemistry, 280: 30158-30165 (2005), American Society for
Biochemistry and Molecular Biology, Inc., 9650 Rockville Pike,
Bethesda, MD 20814 U.S.A. cited by applicant .
Xue et al., "Embryonic Lethality and Vascular Defects in Mice
Lacking the Notch Ligand Jagged1," Human Molecular Genetics, 8:
723-730 (1999), Oxford University Press, McLean, VA 22101-0850,
USA. cited by applicant .
Zagouras et al., "Alterations in Notch Signaling in Neoplastic
Lesions of the Human Cervix," PNAS, 92: 6414-6418 (1995), National
Academy of Sciences, Washington, DC 20001. cited by applicant .
The Extended European Search Report issued in European Application
No. EP 07 777 332.3, on Aug. 11, 2009 (10 pages). cited by
applicant .
Sakamoto, K., et al., "Distinct roles of EGF repeats for the Notch
signaling system," Experimental Cell Research, 2005, 281-291,
302(2), Elsevier, Orlando, FL, XP-004649921. cited by applicant
.
Shao, L., et al., "Fringe modifies O-fucose on mouse Notch1 at
epidermal growth factor-like repeats within the ligand-binding site
and the Abruptex region," The Journal of Biological Chemistry,
2003, 7775-7782, 278(10), American Society for Biochemistry and
Molecular Biology, Bethesda, MD, XP-002538409. cited by applicant
.
Peters, N., et al., "CADASIL-associated Notch3 mutations have
differential effects both on ligand binding and ligand-induced
Notch3 receptor signaling through RBP-Jk," Experimental Cell
Research, 2004, 454-464, 299 (2), Elsevier, Orlando, FL,
XP-004537012. cited by applicant .
Pei, Z. and Baker, N., "Competition between Delta and the Abruptex
domain of Notch," BMC Dev. Biol. 8:4, BioMed Central, England
(2008). cited by applicant .
Luo, B., et al., "Isolation and functional analysis of a cDNA for
human Jagged2, a gene encoding a ligand for the Notch1 receptor,"
Mol. Cell. Biol.17:6057-6067, American Society for Microbiology,
United States (1997). cited by applicant .
International Search Report for International Application No.
PCT/US09/03994, ISA/US, Alexandria, Virgina, USA, mailed on Jul.
23, 2010. cited by applicant .
Shimizu, K., et al., "Physical 1-15 interaction of Delta1, Jagged1,
and Jagged2 with Notch1 and Notch3 receptors," Biochem. Biophys.
Res. Commun. 276:385-389, Academic Press, United States (2000).
cited by applicant .
Rand, M., et al., "Calcium binding to tandem repeats of EGF-like
modules. Expression and characterization of the EGF-like modules of
human Notch-1 implicated in receptor-ligand interactions," Protein
Science 6:2059-2071, Cambridge University Press, United Kingdom
(1997). cited by applicant .
Hambleton, S., et al., "Structural and Functional Properties of the
Human Notch-1 Ligand Binding Region," Structure 12:2173-2183,
Current Biology, Ltd., United States (2004). cited by applicant
.
Miele, L., Gamma-Secretase and Notch Signaling: Novel Therapeutic
Targets In Breast Cancer, DTIC (Online), accessed at
http://www.dtic.mil/cgi-bin/GetTRDoc?Location=U2&doc=GetTRDoc.pdf&AD=ADA4-
46389 (retrieved on Jan. 12, 2010). cited by applicant .
Dikic, I., et al., "Notch: Implications of endogenous inhibitors
for therapy," Bioessays 32:481-487, John Wiley & Sons, United
States (2010). cited by applicant .
Lin, L., et al., "Targeting Specific Regions of the Notch3
Ligand-Binding Domain Induces Apoptosis and Inhibits Tumor Growth
in Lung Cancer," Can. Res. 70:632-638, American Assoc. for Cancer
Research, United States (2010). cited by applicant .
Bheeshmachar, G., et al., "Evidence for a Role for Notch Signaling
in the Cytokine-Dependent Survival of Activated T cells," J.
Immunol. 177:5041-5050, The American Association of Immunologists
Inc., United States (2006). cited by applicant .
Extended European Search Report of European Appl. No. 08 72 4737.5,
European Patent Office, Munich, Germany, dated Sep. 24, 2010. cited
by applicant .
Novus, "Biologicals Product: Mouse Monoclonal anti-Notch 1 (A6)
antibody datasheet," XP008115324, accessed at
http://www.novusbio.com/data.sub.--sheet/pdf.sub.--data.sub.--sheet/5985,
2006. cited by applicant .
Allenspach, E.J., et al., "Notch Signaling in Cancer," Cancer Biol.
1:466-476, Landes Bioscience, United States (2002). cited by
applicant .
Armstrong, F., et al., "NOTCH is a key regulator of human T-cell
acute leukemia initiating cell activity," Blood 113:1730-1740, The
American Society of Hematology, United States (2009). cited by
applicant .
Bellavia, D., et al., "Constitutive activtion of NF-Kband T-cell
leukemia/lymphoma in Notch3 transgenic mice," EMBO J. 19:3337-3348,
Oxford University Press, United States (2000). cited by applicant
.
Callahan, R. & Raafat, A., "Notch Signaling in Mammary Gland
Tumorigenesis," Journal of Mammary Gland Biology and Neoplasia
6:23-36, Plenum Publishing Corporation, United States (2001). cited
by applicant .
Campbell, A.M., "Monoclonal antibody technology," vol. 13, pp.
v-29, Elsevier Science Publishers B.V, The Netherlands, 1984. cited
by applicant .
Cox, C.V., et al., "Characterization of acute lymphoblatic leukemia
progenitor cells," Blood 104:2919-2925, The American Society of
Hematology, United States (2004). cited by applicant .
Deftos, M.L., et al., "Correlating notch signaling with thymocyte
maturation," Immunity 9:777-786, Cell Press, United States (1998).
cited by applicant .
English language Abstract of WIPO Patent Publication No. WO
02/00576 A1, Jan. 3, 2002. cited by applicant .
Fleming, R.J., et al., "The Notch receptor and its ligands," Trends
in Cell Biol. 7:437-441, Elsevier Science Ltd., The Netherlands
(1997). cited by applicant .
Fre, S., et al, "Notch signals control the fate of immature
progenitor cells in the intestine," Nature 435:964-968, Nature
Publishing Group, England (2005). cited by applicant .
Gallahan, D., and Callahan, R., "The mouse mammary tumor associated
gene INT3 is a unique member of the NOTCH gene family
(NOTCH4),"Oncogene 14:1838-1890, Stockton Press, United States
(1997). cited by applicant .
Grabher, C., et al., "Notch 1 activation in the molecular
pathogensis of T-cell acute lymphoblatic leukaemia," Nature Reviews
Cancer 6:347-359, Nature Publishing Group, England (2006). cited by
applicant .
Imatani, A. and Callahan, R., "Identification of a novel
NOTCH-4/INT-3 RNA species encoding an activated gene product in
certain human tumor cell lines," Oncogene 19:223-231, Macmillan
Publishers Ltd., England (2000). cited by applicant .
International Search Report for International Application No.
PCT/US08/00884, United States Patent and Trademark Office, U.S.A.,
mailed on Oct. 1, 2008. cited by applicant .
International Search Report for International Application No.
PCT/US09/03995, United States Patent and Trademark Office, U.S.A.,
mailed on Mar. 2, 2010. cited by applicant .
International Search Report for International Application No.
PCT/US2008/001948, USPTO, mailed on Oct. 15, 2008. cited by
applicant .
Jang, M.S., et al., "Notch signaling as a target in multimodality
cancer therapy," Curr. Opin. Mol. Ther. 2(1):55-65, Thomson Reuters
(Scientific) Ltd., England (Feb. 2000). cited by applicant .
Jarriault, S., et al., "Signaling downstream of activated mammalian
Notch," Nature 377:355-358, Nature Publishing Group, England
(1995). cited by applicant .
Jehn, B.M., et al., "Cutting edge: protective effects of notch-1 on
TCR-induced apoptosis," J. Immunol. 162:635-638, The American
Association of Immunologists, United States (1999). cited by
applicant .
Jundt, F., et al., "Activated Notch1 signaling promotes tumor cell
proliferation and survival in Hodgkin and anaplastic large cell
lymphoma," Blood 99:3398-3403. The American Society of Hematology,
United States (2002). cited by applicant .
Lee, J-S, et al., "Intracisternal Type A Particle-Mediated
Activation of the Notch4/int3 Gene in a Mouse Mammary Tumor:
Generation of Truncated Notch4/int3 mRNAs by Retroviral Splicing
Events." J. Virol. 73:5166-5171, American Society for Microbiology,
United States (1999). cited by applicant .
Lee, S-H, et al., "Mutational analysis of NOTCH1, 2, 3, and 4 genes
in common solid cancers and acute leukemias," APMIS 115:1357-1363,
The Authors Journal Compilation, United States (2007). cited by
applicant .
Li, K., et al., "Modulation of Notch Signaling by Antibodies
Specific for the Extracellular Negative Regulatory Region of
NOTCH3," J. Biol. Chem. 283:8046-8054, The American Society for
Biochemistry and Molecular Biology, Inc., United States (2008).
cited by applicant .
Li, L., et al., "The Human Homolog of Rat Jagged1 Expressed by
Marrow Stroma Inhibits Differentiation of 32D Cells through
Interaction with Notch1," Immunity 8:43-55, Cell PRess, United
States (1998). cited by applicant .
Li, L., et al., "Cloning, Characterization, and the Complete
56.8-Kilobase DNA Sequence of the Human NOTCH4 Gene," Genomics
51:45-48, Academic Press, United States (1998). cited by applicant
.
Lindsell, C.E., et al., "Jagged: A Mammalian Ligand That Activates
Notch1," Cell 80:909-917, Cell Press, United States (1995). cited
by applicant .
Liu, Z., et al., "Notch1 loss of heterozygosity causes vascular
tumors and lethal hemorrhage in mice," J. Clin. Invest.
121(2):800-8, American Society for Clincal Investigation, United
States (Feb. 2011; Epub Jan. 25, 2011). cited by applicant .
Miele, L., & Osborne, B., "Arbiter of Differentiation and
Death: Notch Signaling Meets Apoptosis," J. Cell Physiol.
181:393-409, Wiley-Liss, Inc., United States (1999). cited by
applicant .
Nam, Y., et al., "Notch signaling as a therapeutic target," Curr.
Opin. Chem. Biol. 6:501-509, Elsevier Science Ltd., Holland (2002).
cited by applicant .
Pelegrin, A., et al., "[Immunotargeting of tumors: state of the art
and prospects in 2000]," Bull. Cancer 87(11):777-91, John Libbey
Eurotext, France (Nov. 2000) in the English language. cited by
applicant .
Pelegrin, A., et al., "[Immunotargeting of tumors: state of the art
and prospects in 2000]," Bull. Cancer 87(11):777-91, John Libbey
Eurotext, France (Nov. 2000) in the French language. cited by
applicant .
Sambandam, A., et al., "Notch signaling controls the generation and
differentiation of early T lineage progenitors," Nature Immunol.
6:663-670, Nature Publishing Group, England (2005). cited by
applicant .
Soriano, J.V., et al., "Expression of an activated Notch(int-3)
oncoprotein disrupts morphogenesis and induces and invasive
phenotype in mammary epithelial cells in vitro," Int. J. Cancer 86:
652-659, Wiley-Liss, Inc., United States (2000). cited by applicant
.
Sugaya, K., et al., "Gene organization of human NOTCH4 and (CTG)n
polymorphism in this human counterpart gene of mouse proto-oncogene
Int3," Gene 189:235-244, Elsevier Science B.V., Holland (1997).
cited by applicant .
Summons to attend oral proceedings pursuant to Rule 115(1) EPC for
European Patent Application 05722705.0-2402/1718767, European
Patent Office, Germany, mailed on Feb. 9, 2011. cited by applicant
.
Thelu, J., et al., "Notch signaling is linked to epidermal cell
differentiation level in basal cell carcinoma, psoriasis and wound
healing," BMC Dermatology 2:7, BioMed Central, England (2002).
cited by applicant .
Van Es, J.H., et al., "Notch/.gamma.-secretase inhibition turns
proliferative cells in intestinal crypts and adenomas into goblet
cells," Nature 435:959-963, Nature Publishing Group, England
(2005). cited by applicant .
Weng, A.P., and Aster, J.C., "Multiple niches for Notch in cancer:
context is everything," Curr. Opin. Genet. Dev. 14(1):48-54,
Elsevier, England (Feb. 2004). cited by applicant .
Weng, A.P., et al., "Activating Mutations of NOTCH1 in Human T Cell
Acute Lymphoblastic Leukemia," Science 306:269-271, Nature
Publishing Group, England (2004). cited by applicant .
Weng, A.P., et al., "Growth Suppression of Pre-T Acute
Lymphoblastic Leukemia Cells by Inhibition of Notch Signaling,"
Mol. Cell Biol. 23:655-644, American Society for Microbiology,
United States (2003). cited by applicant .
Axelson, H., "Notch signaling and cancer: emerging complexity,"
Semin. Cancer Biol. 14:317-319, Elsevier Ltd., England (2004).
cited by applicant .
Curry, C.L., et al., "Gamma secretase inhibitor blocks Notch
activation and induces apoptosis in Kaposi's sarcoma tumor cells,"
Oncogene 24:6333-6344, Nature Publishing Group, England (2005).
cited by applicant .
Dontu, G., et al., "Role of Notch signaling in cell-fate
determination of human mammary stem/progenitor cells," Breast
Cancer Res. 6:R605-R615, BioMed Central Ltd., England (2004). cited
by applicant .
Duan, Z., et al., "A Novel Notch Protein, N2N, Targeted by
Neutrophil Elastase and Implicated in Hereditary Neutropenia," Mol.
Cell. Biol. 24(1):58-70, American Society for Microbiology, United
States (2004). cited by applicant .
Harper, J.A , et al., "Notch signaling in development and disease,"
Clin. Genet. 64:461-472, Blackwell Munksgaard, Denmark (2003).
cited by applicant .
Hopfer, O., et al., "The Notch pathway in ovarian carcinomas and
adenomas," Br. J. Cancer 93:709-718, Cancer Research UK, England
(2005). cited by applicant .
Huang, E.Y., et al., "Surface Expression of Notch1 on Thymocytes:
Correlation with the Double-Negative to Double-Positive
Transition," J. Immunol. 171:2296-2304, The American Association of
Immunologists, United States (2003). cited by applicant .
Maillard, I., et al., "Mastermind critically regulates
Notch-mediated lymphoid cell fate decisions," Blood 104:1696-1702,
The American Society of Hematology, United States (2004). cited by
applicant .
Qin, J.-Z., et al., "p53-independent NOXA induction overcomes
apoptotic resistance of malignant melanomas," Mol. Cancer Ther.
3(8):895-902, American Association for Cancer Research, Inc.,
United States (2004). cited by applicant .
Santa Cruz Biotechnology, Inc., "Notch 2 (25-255): sc-5545
datasheet," downloaded on Dec. 2, 2009. cited by applicant .
NCBI Entrez, GenBank Report, Accession No. P01724, Burstein, Y. and
Schechter, I., Entry Date Jul. 21, 1986, last updated Nov. 4, 2008.
cited by applicant .
NCBI Entrez, GenBank Report, Accession No. Q8VDC9, Sembi, P., Entry
Date Mar. 1, 2002, last updated Oct. 31, 2006. cited by applicant
.
International Search Report for International Application No.
PCT/US11/21135, International Searching Authority, Alexandria,
Virginia, United States, mailed Jul. 20, 2011. cited by applicant
.
Written Opinion of the International Searching Authority for
International Application No. PCT/US11/21135, International
Searching Authority, Alexandria, Virginia, United States, mailed
Jul. 20, 2011. cited by applicant .
Liu, H., et al., "Notch3 is Critical for Proper Angiogenesis and
Mural Cell Investment," Circ. Res. 107:860-70, American Heart
Association, United States (2010). cited by applicant .
Bolos, V., et al., "Notch Signaling in Development and Cancer,"
Endocrine Reviews 28(3):339-363, The Endocrine Society, United
States (2007). cited by applicant .
Miele, L., et al., "NOTCH Signaling as a Novel Cancer Therapeutic
Target," Curr. Cancer Drug Targets 6(4):313-323, Bentham Science
Publishers, Ltd., Netherlands (2006). cited by applicant .
Tanaka M., et al., "Asymmetric localization of Notch2 on the
microvillous surface in choroid plexus epithelial cells,"
Histochem. Cell Biol. 127(4):449-56, Epub Jan. 12, 2007, Springer
Verlag, Germany. cited by applicant .
Jurynczyk M., et al., "Notch3 Inhibition in Myelin-Reactive T Cells
Down-Regulates Protein Kinase C.theta.and Attenuates Experimental
Autoimmune Encephalomyelitis," J. Immunology 180(4):2634-40, The
American Association of Immunologists Inc., United States (2008).
cited by applicant .
Bendig, M., "Humanization of Rodent Monoclonal Antibodies by CDR
Grafting," in METHODS: A companion to methods in Enzymology, vol.
8., pp. 83-93 (1995). cited by applicant .
Greenspan, N.S. and Di Cera, E., "Defining epitopes: It's not as
easy as it seems," Nat. Biotechnol. 17(10):936-7, Nature America
Publishing, United States (1999). cited by applicant .
Panka, D.J., et al., "Variable region framework differences result
in decreased or increased affinity of variant anti-digoxin
antibodies," Proc Natl Acad Sci U S A. 85(9):3080-4, National
Academy of Sciences, United States (1988). cited by applicant .
Sriuranpong, V., et al., "Notch Signaling Induces Cell Cycle Arrest
In Small Cell Lung Cancer Cells," Cancer Res. 61(7):3200-5,
American Association for Cancer Research, United States (2001).
cited by applicant .
Talora, C., et al., "Specific down-modulation of Notch1 signaling
in cervical cancer cells is required for sustained HPV-E6/E7
expression and late steps of malignant transformation," Genes Dev.
16(17):2252-63, Cold Spring Harbor Laboratory Press, United States
(2002). cited by applicant .
Ahmad, I., et al., "Involvement of Notch-1 in mammalian retinal
neurogenesis: association of Notch-1 activity with both immature
and terminally differentiated cells," Mech. Dev. 53(1):73-85,
Elsevier Scientific Publishers, Ireland (1995). cited by applicant
.
Houde, C., et al., "Overexpression of the NOTCH ligand JAG2 in
malignant plasma cells from multiple myeloma patients and cell
lines," Blood 104(12):3697-704, American Society of Hematology,
United States (2004). cited by applicant .
Shawber, C., et al., "Notch signaling inhibits muscle cell
differentiation through a CBF1-independent pathway," Development
122(12):3765-73, Company of Biologists Limited, England (1996).
cited by applicant .
"4G1" Notch1 monoclonal antibody; Abnova technical datasheet,
accessed at
www.abnova.com/products/products.sub.--details.asp?Catalog.sub.--id=1-1-0-
0004851-M10, accessed on Nov. 9, 2012; 8 total pages. cited by
applicant .
McDaniell, R., et al., "NOTCH2 Mutations Cause Alagille Syndrome, a
Heterogenous Disorder of the Notch Signaling Pathway," Am. J. Hum.
Genet.79(1):169-73, University of Chicago Press, United States
(2006). cited by applicant .
Nickoloff, B.J., et al., "Notch signaling as a therapeutic target
in cancer: a new approach to the development of cell fate modifying
agents," Oncogene 22(42):6598-608, Nature Publishing Group, England
(2003). cited by applicant .
Varnum-Finney, B., et al., "The Notch Ligand, Jagged-1, Influences
the Development of Primitive Hematopoietic Precursor Cells," Blood
91(11):4084-91, The American Society of Hematology, United States
(1998). cited by applicant .
Jundt, F., et al., "Jagged1-induced Notch signaling drives
proliferation of multiple myeloma cells," Blood 103:3511-3515, The
American Society of Hematology, United States (2004). cited by
applicant .
Wu, Y., "Therapeutic antibody targeting of individual Notch
receptors," Nature 464:1052-1057, Nature Publishing Group, England
(2010). cited by applicant .
Siebel, C.W. "PL07-04 Notch Antibody Antagonists for Cancer
Therapy," Invited Abstracts (Plenary Session), Abstract nr PL07-04,
American Association for Cancer Research, United States (2007).
cited by applicant .
Gordon, W.R., et al., "Structural basis for autoinhibition of
Notch," Nat. Struct. Mol. Biol. 14(4):295-300, Nature Pub. Group,
United States (2007). cited by applicant .
Sanchez-Irizarry, C., "Notch Subunit Heterodimerization and
Prevention of Ligand-independent Proteolytic Activation Depend,
Respectively, on a Novel Domain and the LNR Repeats," Mol. Cell
Biol. 24(21):9265-9273, American Society for Microbiology, United
States (2004). cited by applicant .
Roy, M., et al., "The multifaced role of Notch in cancer," Curr.
Opin. Genet. Dev. 17(1):52-59, Elsevier, England (2007). cited by
applicant .
Supplemental Data for Li, K., et al., "Modulation of Notch
Signaling by Antibodies Specific for the Extracellular Negative
Regulatory Region of NOTCH3," J. Biol. Chem. 283:8046-8054, The
American Society for Biochemistry and Molecular Biology, Inc.,
United States (2008); 9 total pages. cited by applicant.
|
Primary Examiner: Bunner; Bridget E
Attorney, Agent or Firm: Sterne Kessler Goldstein & Fox
P.L.L.C.
Claims
What is claimed is:
1. An isolated antibody that specifically binds human Notch1, which
comprises: (a) a heavy chain CDR1 comprising RGYWIE (SEQ ID NO:15);
a heavy chain CDR2 comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16); and
a heavy chain CDR3 comprising FDGNYGYYAMDY (SEQ ID NO:17); and (b)
a light chain CDR1 comprising RSSTGAVTTSNYAN (SEQ ID NO:18); a
light chain CDR2 comprising GTNNRAP (SEQ ID NO:19); and a light
chain CDR3 comprising ALWYSNHWVFGGGTKL (SEQ ID NO:20).
2. The antibody of claim 1, which binds to a non-ligand binding
membrane proximal region of an extracellular domain of human Notch1
receptor.
3. The antibody of claim 2, wherein the non-ligand binding membrane
proximal region of an extracellular domain of human Notch1 receptor
comprises SEQ ID NO:2.
4. The isolated antibody of claim 1, wherein the antibody
comprises: (a) a heavy chain variable region having at least 90%
sequence identity to SEQ ID NO:14 or SEQ ID NO:24; and/or (b) a
light chain variable region having at least 90% sequence identity
to SEQ ID NO:8, SEQ ID NO:28, or SEQ ID NO:32.
5. The antibody of claim 1, which comprises: (a) a heavy chain
variable region comprising SEQ ID NO:24; and (b) a light chain
variable region comprising SEQ ID NO:28.
6. The antibody of claim 1, which is a recombinant antibody, a
monoclonal antibody, a chimeric antibody, a humanized antibody, a
human antibody, an antibody fragment, a bispecific antibody, a
monospecific antibody, a monovalent antibody, an IgG1 antibody, or
an IgG2 antibody.
7. A pharmaceutical composition comprising the antibody of claim
1.
8. A cell line producing the antibody of claim 1.
9. An isolated polynucleotide molecule comprising a polynucleotide
that encodes the antibody of claim 1.
10. A method of inhibiting growth of a tumor or a tumor cell,
comprising contacting the tumor or tumor cell with an effective
amount of the antibody of claim 1.
11. The method of claim 10, wherein the tumor is selected from the
group consisting of a breast tumor, colorectal tumor, hepatic
tumor, renal tumor, lung tumor, pancreatic tumor, ovarian tumor,
prostate tumor, and head and neck tumor.
12. A method of reducing tumorigenicity of a tumor in a subject,
comprising administering to the subject a therapeutically effective
amount of the antibody of claim 1, wherein the tumor comprises
cancer stem cells and the frequency of the cancer stem cells in the
tumor is reduced by administration of the antibody.
13. The antibody of claim 1, which comprises: (a) a heavy chain
variable region comprising SEQ ID NO:24; and (b) a light chain
variable region comprising SEQ ID NO:32.
14. The antibody of claim 1, which is a monoclonal antibody.
15. The antibody of claim 14, which is a humanized antibody.
16. The antibody of claim 1, which is 52M51H4L3.
17. An isolated monoclonal antibody comprising heavy and light
chain variable regions identical in sequence to the heavy and light
chain variable regions encoded by the plasmid on deposit as ATCC
Patent Deposit Designation PTA-9549.
18. A method of treating cancer in a subject, comprising
administering to the subject a therapeutically effective amount of
an antibody that specifically binds human Notch1, wherein the
antibody comprises: (a) a heavy chain CDR1 comprising RGYWIE (SEQ
ID NO:15), a heavy chain CDR2 comprising QILPGTGRTNYNEKFKG (SEQ ID
NO:16), and a heavy chain CDR3 comprising FDGNYGYYAMDY (SEQ ID
NO:17); (b) a light chain CDR1 comprising RSSTGAVTTSNYAN (SEQ ID
NO:18), a light chain CDR2 comprising GTNNRAP (SEQ ID NO:19), and a
light chain CDR3 comprising ALWYSNHWVFGGGTKL (SEQ ID NO:20).
19. The method of claim 18, wherein the cancer is selected from the
group consisting of a breast cancer, colorectal cancer, hepatic
cancer, kidney cancer, liver cancer, lung cancer, pancreatic
cancer, gastrointestinal cancer, melanoma, ovarian cancer, prostate
cancer, cervical cancer, bladder cancer, glioblastoma, and head and
neck cancer.
20. The method of claim 18, further comprising administering to the
subject at least one additional anti-cancer or therapeutic
agent.
21. The method of claim 20, wherein the additional therapeutic
agent is a chemotherapeutic agent.
22. The method of claim 18, wherein the antibody is 52M51H4L3.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to compositions comprising an agent
that binds a human Notch receptor and methods of using the
compositions for characterizing, diagnosing, and treating cancer
and other diseases. In particular, the present invention provides
antibodies that specifically bind to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor and inhibit tumor growth. The present invention further
provides methods of treating cancer, the methods comprising
administering a therapeutically effective amount of an antibody
that specifically binds to a non-ligand binding membrane proximal
region of the extracellular domain of a human Notch1 receptor
protein and inhibits tumor growth.
2. Background
Cancer is one of the leading causes of death in the developed
world, resulting in over 500,000 deaths per year in the United
States alone. Over one million people are diagnosed with cancer in
the U.S. each year, and overall it is estimated that more than 1 in
3 people will develop some form of cancer during their lifetime.
Though there are more than 200 different types of cancer, four of
them--breast, lung, colorectal, and prostate--account for over half
of all new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).
Cancer arises from dysregulation of the mechanisms that control
normal tissue development and maintenance, and increasingly stem
cells are thought to play a central role (Beachy et al., 2004,
Nature 432:324). During normal animal development, cells of most or
all tissues are derived from normal precursors, called stem cells
(Morrison et al., 1997, Cell 88:287-98; Morrison et al., 1997,
Curr. Opin. Immunol. 9:216-21; Morrison et al., 1995, Annu. Rev.
Cell. Dev. Biol. 11:35-71). Stem cells are cells that: (1) have
extensive proliferative capacity; 2) are capable of asymmetric cell
division to generate one or more kinds of progeny with reduced
proliferative and/or developmental potential; and (3) are capable
of symmetric cell divisions for self-renewal or self-maintenance.
The best-known example of adult cell renewal by the differentiation
of stem cells is the hematopoietic system where developmentally
immature precursors (hematopoietic stem and progenitor cells)
respond to molecular signals to form the varied blood and lymphoid
cell types. Other cells, including cells of the gut, breast ductal
system, and skin are constantly replenished from a small population
of stem cells in each tissue, and recent studies suggest that most
other adult tissues also harbor stem cells, including the
brain.
Solid tumors are composed of heterogeneous cell populations. For
example, breast cancers are a mixture of cancer cells and normal
cells, including mesenchymal (stromal) cells, inflammatory cells,
and endothelial cells. Classic models of cancer hold that
phenotypically distinct cancer cell populations all have the
capacity to proliferate and give rise to a new tumor. In the
classical model, tumor cell heterogeneity results from
environmental factors as well as ongoing mutations within cancer
cells resulting in a diverse population of tumorigenic cells. This
model rests on the idea that all populations of tumor cells would
have some degree of tumorigenic potential. (Pandis et al., 1998,
Genes, Chromosomes & Cancer 12:122-129; Kuukasjrvi et al.,
1997, Cancer Res. 57:1597-1604; Bonsing et al., 1993, Cancer
71:382-391; Bonsing et al., 2000, Genes Chromosomes & Cancer
82: 173-183; Beerman H et al., 1991, Cytometry 12:147-54; Aubele M
& Werner M, 1999, Analyt. Cell. Path. 19:53; Shen L et al.,
2000, Cancer Res. 60:3884).
An alternative model for the observed solid tumor cell
heterogeneity is that solid tumors result from a "solid tumor stem
cell" (or "cancer stem cell" from a solid tumor) that subsequently
undergoes chaotic development through both symmetric and asymmetric
rounds of cell divisions. In this stem cell model, solid tumors
contain a distinct and limited (possibly even rare) subset of cells
that share the properties of normal "stem cells", in that they
extensively proliferate and efficiently give rise both to
additional solid tumor stem cells (self-renewal) and to the
majority of tumor cells of a solid tumor that lack tumorigenic
potential. Indeed, mutations within a long-lived stem cell
population may initiate the formation of cancer stem cells that
underlie the growth and maintenance of tumors and whose presence
contributes to the failure of current therapeutic approaches.
The stem cell nature of cancer was first revealed in the blood
cancer, acute myeloid leukemia (AML) (Lapidot et al., 1994, Nature
367:645-8). More recently it has been demonstrated that malignant
human breast tumors similarly harbor a small, distinct population
of cancer stem cells enriched for the ability to form tumors in
immunodeficient mice. An ESA+, CD44+, CD24-/low, Lin-cell
population was found to be 50-fold enriched for tumorigenic cells
compared to unfractionated tumor cells (Al-Hajj et al., 2003, PNAS
100:3983-8). The ability to prospectively isolate the tumorigenic
cancer cells has permitted investigation of critical biological
pathways that underlie tumorigenicity in these cells, and thus
promises the development of better diagnostic assays and
therapeutics for cancer patients. It is toward this purpose that
this invention is directed.
Normal stem cells and cancer stem cells share the ability to
proliferate and self-renew, thus it is not surprising that a number
of genes that regulate normal stem cell development contribute to
tumorigenesis (reviewed in Reya et al., 2001, Nature 414:105-111
and Taipale & Beachy, 2001, Nature 411:349-354). The present
invention identifies Notch receptor, for example, Notch1, as a
marker of cancer stem cells, implicating the Notch signaling
pathway in the maintenance of cancer stem cells and as a target for
treating cancer via the elimination of these tumorigenic cells.
The Notch signaling pathway is one of several critical regulators
of embryonic pattern formation, post-embryonic tissue maintenance,
and stem cell biology. More specifically, Notch signaling is
involved in the process of lateral inhibition between adjacent cell
fates and plays an important role in cell fate determination during
asymmetric cell divisions. Unregulated Notch signaling is
associated with numerous human cancers where it can alter the
developmental fate of tumor cells to maintain them in an
undifferentiated and proliferative state (Brennan and Brown, 2003,
Breast Cancer Res. 5:69). Thus carcinogenesis can proceed by
usurping homeostatic mechanisms controlling normal development and
tissue repair by stem cell populations (Beachy et al., 2004, Nature
432:324).
The Notch receptor was first identified in Drosophila mutants with
haploinsufficiency resulting in notches at the wing margin, whereas
loss-of-function produces an embryonic lethal "neurogenic"
phenotype where cells of the epidermis switch fate to neural tissue
(Moohr, 1919, Genet. 4:252; Poulson, 1937, PNAS 23:133; Poulson,
1940, J. Exp. Zool. 83:271). The Notch receptor is a single-pass
transmembrane receptor containing numerous tandem epidermal growth
factor (EGF)-like repeats and three cysteine-rich Notch/LIN-12
repeats (LNRs) within a large extracellular domain (Wharton et al.,
1985, Cell 43:567; Kidd et al., 1986, Mol. Cell Biol. 6:3094;
reviewed in Artavanis et al., 1999, Science 284:770). The LNRs and
an additional C-terminal tail of approximately 103 amino acids of
the extracellular domain are referred to herein as the "membrane
proximal region". This region is also known as, and referred to as
the Notch negative regulatory region (NRR).
Mammalian Notch receptors undergo cleavage to both form the mature
receptor and following ligand binding to activate downstream
signaling. A furin-like protease cleaves the Notch receptor
precursors during maturation to generate juxtamembrane heterodimers
that comprise a non-covalently associated extracellular subunit and
a transmembrane subunit held together in an auto-inhibitory state.
Ligand binding relieves this inhibition and induces cleavage of the
Notch receptor by an ADAM-type metalloprotease and gamma-secretase,
the latter of which releases the intracellular domain (ICD) into
the cytoplasm, allowing it to translocate into the nucleus to
activate gene transcription. Cleavage by ADAM occurs within the
non-ligand binding cleavage domain within the juxtamembrane
negative regulatory region (NRR) (See FIG. 1A). In the Notch1
receptor this region encompasses from about amino acid 1427 to
about amino acid 1732.
Four mammalian Notch proteins have been identified (Notch1, Notch2,
Notch3, and Notch4), and mutations in these receptors invariably
result in developmental abnormalities and human pathologies
including several cancers as described in detail below (Gridley,
1997, Mol. Cell Neurosci. 9:103; Joutel & Tournier-Lasserve,
1998, Semin. Cell Dev. Biol. 9:619-25).
The Notch receptor is activated by single-pass transmembrane
ligands of the Delta, Serrated, Lag-2 (DSL) family. There are five
known Notch ligands in mammals: Delta-like 1 (DLL1), Delta-like 3
(DLL3), Delta-like 4 (DLL4), Jagged 1 and Jagged 2 characterized by
a DSL domain and tandem EGF-like repeats within the extracellular
domain. The extracellular domain of the Notch receptor interacts
with that of its ligands, typically on adjacent cells, resulting in
two proteolytic cleavages of Notch; one extracellular cleavage
mediated by an ADAM (A Disintegrin And Metallopeptidase) protease
and one cleavage within the transmembrane domain mediated by gamma
secretase. This latter cleavage generates the Notch intracellular
domain (ICD), which then enters the nucleus where it activates the
CBF1, Suppressor of Hairless [Su(H)], Lag-2 (CSL) family of
transcription factors as the major downstream effectors to increase
transcription of nuclear basic helix-loop-helix transcription
factors of the Hairy and Enhancer of Split [E(spl)] family
(Artavanis et al., 1999, Science 284:770; Brennan and Brown, 2003,
Breast Cancer Res. 5:69; Iso et al., 2003, Arterioscler. Thromb.
Vasc. Biol. 23:543). Alternative intracellular pathways involving
the cytoplasmic protein Deltex identified in Drosophila may also
exist in mammals (Martinez et al., 2002, Curr. Opin. Genet. Dev.
12:524-33), and this Deltex-dependent pathway may act to suppress
expression of Wnt target genes (Brennan et al., 1999, Curr. Biol.
9:707-710; Lawrence et al., 2001, Curr. Biol. 11:375-85).
Hematopoietic stem cells (HSCs) are the best understood stem cells
in the body, and Notch signaling is implicated both in their normal
maintenance as well as in leukemic transformation (Kopper &
Hajdu, 2004, Pathol. Oncol. Res. 10:69-73). HSCs are a rare
population of cells that reside in a stromal niche within the adult
bone marrow. These cells are characterized both by a unique gene
expression profile as well as an ability to continuously give rise
to more differentiated progenitor cells to reconstitute the entire
hematopoietic system. Constitutive activation of Notch1 signaling
in HSCs and progenitor cells establishes immortalized cell lines
that generate both lymphoid and myeloid cells in vitro and in
long-term reconstitution assays (Varnum-Finney et al., 2000, Nat.
Med. 6:1278-81), and the presence of Jagged1 increases engraftment
of human bone marrow cell populations enriched for HSCs (Karanu et
al., 2000, J. Exp. Med. 192:1365-72). More recently, Notch
signaling has been demonstrated in HSCs in vivo and shown to be
involved in inhibiting HSC differentiation. Furthermore, Notch
signaling appears to be required for Wnt-mediated HSC self-renewal
(Duncan et al., 2005, Nat. Immunol. 6:314).
The Notch signaling pathway also plays a central role in the
maintenance of neural stem cells and is implicated both in their
normal maintenance as well as in brain cancers (Kopper & Hajdu,
2004, Pathol. Oncol. Res. 10:69-73; Purow et al., 2005, Cancer Res.
65:2353-63; Hallahan et al., 2004, Cancer Res. 64:7794-800). Neural
stem cells give rise to all neuronal and glial cells in the
mammalian nervous system during development, and more recently have
been identified in the adult brain (Gage, 2000, Science
287:1433-8). Mice deficient for Notch1; the Notch target genes
Hes1, 3, and 5; and a regulator of Notch signaling presenilin1
(PS1) show decreased numbers of embryonic neural stem cells.
Furthermore, adult neural stem cells are reduced in the brains of
PS1 heterozygote mice (Nakamura et al., 2000, J. Neurosci.
20:283-93; Hitoshi et al., 2002, Genes Dev. 16:846-58). The
reduction in neural stem cells appears to result from their
premature differentiation into neurons (Hatakeyama et al., 2004,
Dev. 131:5539-50) suggesting that Notch signaling regulates neural
stem cell differentiation and self-renewal.
Aberrant Notch signaling is implicated in a number of human
cancers. The Notch1 gene in humans was first identified in a subset
of T-cell acute lymphoblastic leukemias as a translocated locus
resulting in activation of the Notch pathway (Ellisen et al., 1991,
Cell 66:649-61). Constitutive activation of Notch1 signaling in
T-cells in mouse models similarly generates T-cell lymphomas
suggesting a causative role (Robey et al., 1996, Cell 87:483-92;
Pear et al., 1996, J. Exp. Med. 183:2283-91; Yan et al., 2001,
Blood 98:3793-9; Bellavia et al., 2000, EMBO J. 19:3337-48).
Recently Notch1 point mutations, insertions, and deletions
producing aberrant Notch1 signaling have been found to be
frequently present in both childhood and adult T-cell acute
lymphoblastic leukemia/lymphoma (Pear & Aster, 2004, Curr.
Opin. Hematol. 11:416-33).
The frequent insertion of the mouse mammary tumor virus into both
the Notch1 and Notch4 locus in mammary tumors and the resulting
activated Notch protein fragments first implicated Notch signaling
in breast cancer (Gallahan & Callahan, 1987, J. Virol.
61:66-74; Brennan & Brown, 2003, Breast Cancer Res. 5:69;
Politi et al., 2004, Semin. Cancer Biol. 14:341-7). Further studies
in transgenic mice have confirmed a role for Notch in ductal
branching during normal mammary gland development, and a
constitutively active form of Notch4 in mammary epithelial cells
inhibits epithelial differentiation and results in tumorigenesis
(Jhappan et al., 1992, Genes & Dev. 6:345-5; Gallahan et al.,
1996, Cancer Res. 56:1775-85; Smith et al., 1995, Cell Growth
Differ. 6:563-77; Soriano et al., 2000, Int. J. Cancer 86:652-9;
Uyttendaele et al., 1998, Dev. Biol. 196:204-17; Politi et al.,
2004, Semin. Cancer Biol. 14:341-7). Currently the evidence for a
role for Notch in human breast cancer is limited to the expression
of Notch receptors in breast carcinomas and their correlation with
clinical outcome (Weijzen et al., 2002, Nat. Med. 8:979-86; Parr et
al., 2004, Int. J. Mol. Med. 14:779-86). Furthermore,
overexpression of the Notch pathway has been observed in cervical
cancers (Zagouras et al., 1995, PNAS 92:6414-8), renal cell
carcinomas (Rae et al., 2000, Int. J. Cancer 88:726-32), head and
neck squamous cell carcinomas (Leethanakul et al., 2000, Oncogene
19:3220-4), endometrial cancers (Suzuki et al., 2000, Int. J.
Oncol. 17:1131-9), and neuroblastomas (van Limpt et al., 2000, Med.
Pediatr. Oncol. 35:554-8) suggestive of a potential role for Notch
in the development of a number of neoplasms. Interestingly, Notch
signaling might play a role in the maintenance of the
undifferentiated state of Apc-mutant neoplastic cells of the colon
(van Es & Clevers, 2005, Trends in Mol. Med. 11:496-502).
The Notch pathway is also involved in multiple aspects of vascular
development including proliferation, migration, smooth muscle
differentiation, angiogenesis and arterial-venous differentiation
(Iso et al., 2003, Arterioscler. Thromb. Vasc. Biol. 23:543). For
example, homozygous null mutations in Notch1/4 and Jagged1 as well
as heterozygous loss of DLL4 result in severe though variable
defects in arterial development and yolk sac vascularization.
Furthermore, DLL1-deficient and Notch-2-hypomorphic mice embryos
show hemorrhage that likely results from poor development of
vascular structures (Gale et al., 2004, PNAS, 101:15949-54; Krebs
et al., 2000, Genes Dev. 14:1343-52; Xue et al., 1999, Hum. Mel
Genet. 8:723-30; Hrabe de Angelis et al., 1997, Nature 386:717-21;
McCright et al., 2001, Dev. 128:491-502). In human, mutations in
Jagged1 are associated with Alagille syndrome, a developmental
disorder that includes vascular defects, and mutations in Notch3
are responsible for an inherited vascular dementia (Cadasil) in
which vessel homeostasis is defective (Joutel et al., 1996, Nature
383:707-10).
The identification of Notch1, Notch4, DLL1 and DLL4 as genes
expressed in cancer stem cells compared to normal breast epithelium
suggests that targeting the Notch pathway can help eliminate not
only the majority of nontumorigenic cancer cells, but the
tumorigenic cells responsible for the formation and reoccurrence of
solid tumors. Furthermore, because of the prominent role of
angiogenesis in tumor formation and maintenance, targeting the
Notch pathway can also effectively inhibit angiogenesis, starving a
cancer of nutrients and contributing to its elimination.
Anti-Notch antibodies and their possible use as anti-cancer
therapeutics have been reported. See, e.g., U.S. Patent Application
Publication Nos. 2008/0131434 and 2009/0081238, each of which is
incorporated by reference herein in its entirety. See also
International Publication Nos. WO 2008/057144, WO 2008/076960, and
WO 2008/50525.
BRIEF SUMMARY OF THE INVENTION
The present invention provides agents that bind to a non-ligand
binding membrane proximal region of the extracellular domain of a
Notch1 receptor and compositions, such as pharmaceutical
compositions, comprising those agents. The invention further
provides methods of targeting cancer stem cells with the agents. In
some embodiments, the methods comprise reducing the frequency of
cancer stem cells in a tumor, reducing the number of cancer stem
cells in a tumor, reducing the tumorigenicity of a tumor, and/or
reducing the tumorigenicity of a tumor by reducing the number or
frequency of cancer stem cells in the tumor. The invention also
provides methods of using the agents in the treatment of cancer
and/or in the inhibition of tumor growth.
In one aspect, the invention provides an antibody that specifically
binds to a non-ligand binding membrane proximal region of the
extracellular domain of a Notch1 receptor (e.g., human Notch1). In
some embodiments, the non-ligand binding membrane proximal region
of a Notch1 receptor comprises about amino acid 1427 to about amino
acid 1732 of a human Notch1 receptor. In some embodiments, the
membrane proximal region of a Notch1 receptor comprises SEQ ID
NO:2. In certain embodiments, the antibody specifically binds to a
non-ligand binding membrane proximal region of the extracellular
domain of at least one additional Notch receptor family member.
In some embodiments the antibody is an antagonist of Notch1. In
some embodiments, the antibody inhibits signaling by or activation
of the Notch1 receptor. In some embodiments, the antibody inhibits
Notch1 activity. In some embodiments, the antibody inhibits
cleavage within the membrane proximal region. In certain
embodiments, the antibody inhibits cleavage of the Notch1 receptor
(e.g., cleavage at the S2 site by a metalloprotease) and/or
inhibits activation of the Notch1 receptor by ligand binding. In
some embodiments, the antibody inhibits release or formation of the
intracellular domain (ICD) of Notch1. In certain embodiments, the
antibody inhibits tumor growth.
In certain embodiments, the invention provides an antibody that
binds a non-ligand binding membrane proximal region of the
extracellular domain of a human Notch1 and comprises a heavy chain
CDR1 comprising RGYWIE (SEQ ID NO:15), a heavy chain CDR2
comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16), and/or a heavy chain
CDR3 comprising FDGNYGYYAMDY (SEQ ID NO:17); and/or (b) a light
chain CDR1 comprising RSSTGAVTTSNYAN (SEQ ID NO:18), a light chain
CDR2 comprising GTNNRAP (SEQ ID NO:19), and/or a light chain CDR3
comprising ALWYSNHWVFGGGTKL (SEQ ID NO:20). In some embodiments,
the antibody comprises a heavy chain variable region comprising:
(a) a heavy chain CDR1 comprising RGYWIE (SEQ ID NO:15), or a
variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;
(b) a heavy chain CDR2 comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16),
or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; and/or (c) a heavy chain CDR3 comprising
FDGNYGYYAMDY (SEQ ID NO:17), or a variant thereof comprising 1, 2,
3, or 4 amino acid substitutions. In certain other embodiments, the
antibody comprises (or further comprises) a light chain variable
region comprising: (a) a light chain CDR1 comprising RSSTGAVTTSNYAN
(SEQ ID NO:18), or a variant thereof comprising 1, 2, 3, or 4 amino
acid substitutions; (b) a light chain CDR2 comprising GTNNRAP(SEQ
ID NO:19), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; and/or (c) a light chain CDR3 comprising
ALWYSNHWVFGGGTKL (SEQ ID NO:20), or a variant thereof comprising 1,
2, 3, or 4 amino acid substitutions. In some embodiments, the amino
acid substitutions are conservative amino acid substitutions.
In some embodiments, the invention provides an antibody, 52M51,
produced by the hybridoma cell line deposited with the American
Type Culture Collection (ATCC), 10801 University Boulevard,
Manassas, Va., USA, under the conditions of the Budapest Treaty on
Aug. 7, 2008, and assigned designation number PTA-9405. In some
embodiments, the invention provides a humanized version of antibody
52M51, 52M51H4L3, as encoded by the DNA deposited with the ATCC,
under the conditions of the Budapest Treaty on Oct. 15, 2008, and
assigned designation number PTA-9549. In some embodiments, the
invention provides an antibody that binds to the same epitope as
the epitope to which antibody 52M51 binds.
In another aspect, the invention provides an antibody that binds a
non-ligand binding membrane proximal region of the extracellular
domain of a human Notch1 and the antibody comprises, consists, or
consists essentially of an antibody "52R43" as encoded by the DNA
deposited with the ATCC under the conditions of the Budapest Treaty
on Oct. 15, 2008, and assigned designation number PTA-9548. In some
embodiments, the invention provides an antibody that competes with
52R43 for specific binding to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1.
Pharmaceutical compositions comprising 52R43 and methods of
treating cancer comprising administering therapeutically effective
amounts of the 52R43 antibody are also provided.
In certain embodiments, the invention provides an antibody that
competes with any of the antibodies as described in the
aforementioned embodiments and/or aspects, as well as other
aspects/embodiments described elsewhere herein, for specific
binding to a non-ligand binding membrane proximal region of the
extracellular domain of a human Notch1 (e.g., in a competitive
binding assay). Pharmaceutical compositions comprising the
antibodies described herein and methods of treating cancer
comprising administering therapeutically effective amounts of the
antibodies are also provided.
In certain embodiments of each of the aforementioned aspects or
embodiments, as well as other aspects and/or embodiments described
elsewhere herein, the antibody is a recombinant antibody. In some
embodiments, the antibody is a monoclonal antibody, achimeric
antibody, a humanized antibody, or a human antibody. In certain
embodiments, the antibody is an antibody fragment. In certain
embodiments, the antibody or antibody fragment is monovalent,
monospecific, bivalent, bispecific, or multispecific. In certain
embodiments, the antibody is conjugated to a cytotoxic moiety. In
certain embodiments, the antibody is isolated. In still further
embodiments, the antibody is substantially pure.
Pharmaceutical compositions comprising the antibodies described
herein and methods of treating cancer comprising administering
therapeutically effective amounts of the antibodies described
herein are also provided. In certain embodiments, the
pharmaceutical compositions further comprise a pharmaceutically
acceptable carrier.
In another aspect, the invention provides a polypeptide. In some
embodiments, the polypeptide is an antibody (e.g., an antibody that
specifically binds Notch1), a heavy chain or light chain of an
antibody, and/or a fragment of an antibody. In some embodiments,
the polypeptide is isolated. In certain embodiments, the
polypeptide is substantially pure. In some embodiments, the
polypeptide comprises an amino acid sequence of SEQ ID NO:8, SEQ ID
NO:14, SEQ ID NO:24, SEQ ID NO:28, or SEQ ID NO:32. In some
embodiments, the polypeptide comprises an amino acid sequence of
SEQ ID NO:14 or SEQ ID NO:24 and/or an amino acid sequence of SEQ
ID NO:8, SEQ ID NO:28, or SEQ ID NO:32. In some embodiments, the
polypeptide comprises at least a portion of the amino acid sequence
of SEQ ID NO:14 or SEQ ID NO:24, and/or at least a portion of the
amino acid sequence of SEQ ID NO:8, SEQ ID NO:28, or SEQ ID NO:32.
Pharmaceutical compositions comprising both the polypeptide and a
pharmaceutically acceptable vehicle are further provided, as are
cell lines that produce the polypeptide.
In some embodiments, the polypeptide comprises: (a) a polypeptide
having at least about 80% sequence identity to SEQ ID NO:14 or SEQ
ID NO:24; and/or (b) a polypeptide having at least about 80%
sequence identity to SEQ ID NO:8, SEQ ID NO:28 or SEQ ID NO:32. In
certain embodiments, the polypeptide is an antibody (e.g., an
antibody that specifically binds to the non-ligand binding membrane
proximal region of an extracellular domain of human Notch1). In
certain embodiments, the polypeptide comprises a polypeptide having
at least about 85%, at least about 90%, at least about 95%, at
least about 98%, or about 100% sequence identity to SEQ ID NO:14,
SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:28 or SEQ ID NO:32. In certain
embodiments, the polypeptide comprises a heavy chain variable
region and/or a light chain variable region of the 52M51 antibody.
In some embodiments, the polypeptide comprises a heavy chain
variable region and/or a light chain variable region of a humanized
52M51 antibody. In some embodiment, the polypeptide comprises a
heavy chain variable region and/or a light chain variable region of
antibody 52R43.
In another aspect, the invention provides a polynucleotide molecule
encoding any of the antibodies and/or polypeptides of the
aforementioned aspects, as well as other aspects/embodiments as
described herein. In some embodiments, an expression vector
comprises the polynucleotide molecule. In other embodiments, a host
cell comprises the expression vector. In some embodiments, a host
cell comprises the polynucleotide molecule. In some embodiments,
the host cell is cell line or a hybridoma cell line. In certain
embodiments, the hybridoma cell line produces the 52M51 antibody or
a humanized 52M51 antibody.
In a further aspect, the invention provides a method of inhibiting
activity of Notch1 in a cell, comprising contacting the cell with
an effective amount of any of the antibodies or polypeptides
described in the aforementioned aspects and embodiments, as well as
other aspects/embodiments described elsewhere herein. In certain
embodiments, the cell is a tumor cell.
In another aspect, the invention provides a method of inhibiting
the growth of a tumor in a subject, the method comprising
administering to the subject a therapeutically effective amount of
any of the antibodies or polypeptides described in the
aforementioned aspects and embodiments, as well as other
aspects/embodiments described elsewhere herein. In some
embodiments, the tumor comprises cancer stem cells. In some
embodiments, the methods comprise targeting the cancer stem cells
with the antibodies. In certain embodiments, the methods comprise
reducing the frequency of cancer stem cells in a tumor, reducing
the number of cancer stem cells in a tumor, reducing the
tumorigenicity of a tumor, and/or reducing the tumorigenicity of a
tumor by reducing the number or frequency of cancer stem cells in
the tumor. In some embodiments, the methods comprise inhibiting the
activity of a Notch1 receptor and/or inhibiting growth of a tumor.
In certain embodiments, the tumor is selected from the group
consisting of a breast tumor, colorectal tumor, hepatic tumor,
renal tumor, lung tumor, pancreatic tumor, ovarian tumor, prostate
tumor and head and neck tumor.
In another aspect, the present invention provides methods of
treating cancer in a subject. In some embodiments, the method
comprises administering to a subject a therapeutically effective
amount of any of the antibodies or polypeptides described in the
aforementioned aspects and/or embodiments, as well as other
aspects/embodiments described elsewhere herein. In some
embodiments, the cancer to be treated is breast cancer, colorectal
cancer, hepatic cancer, kidney cancer, liver cancer, lung cancer,
pancreatic cancer, gastrointestinal cancer, melanoma, ovarian
cancer, prostate cancer, cervical cancer, bladder cancer,
glioblastoma, and head and neck cancer. In certain embodiments of
each of the aforementioned aspects or embodiments, as well as other
aspects and/or embodiments described elsewhere herein, the method
of treating cancer comprises inhibiting tumor growth.
In an additional aspect, the invention provides a method of
inhibiting growth of a tumor in a subject, the method comprising
administering to the subject a therapeutically effective amount of
an antibody that specifically binds to a non-ligand binding
membrane proximal region of an extracellular domain of human
Notch1, wherein binding inhibits activity of Notch1.
In a further aspect, the invention provides a method of reducing
the tumorigenicity of a tumor that comprises cancer stem cells by
reducing the frequency or number of cancer stem cells in the tumor,
the method comprising contacting the tumor with an effective amount
of an antibody that inhibits the activity of Notch1.
In certain embodiments of each of the aforementioned aspects and/or
embodiments, as well as other aspects or embodiments described
herein, the methods further comprise administering to the subject
at least one additional anti-cancer and/or therapeutic agent. In
certain embodiments of each of the aforementioned aspects or
embodiments, as well as other aspects and/or embodiments described
elsewhere herein, the antibody or polypeptide is administered to a
subject in combination with an additional treatment for cancer. In
certain embodiments, the additional treatment for cancer comprises
radiation therapy, chemotherapy, and/or an additional antibody
therapeutic. In certain embodiments, the chemotherapy comprises
taxol, irinotecan, gemcitabine and/or oxaliplatin. In certain
embodiments, the additional antibody therapeutic is an antibody
that specifically binds a second human Notch receptor (e.g.,
Notch2) or a human Notch receptor ligand (e.g., DLL4 or JAG1). In
certain embodiments, the additional antibody therapeutic is an
antibody that specifically binds VEGF. In certain embodiments, the
subject treated is a human.
The invention further provides a method of treating cancer in a
human, wherein the cancer comprising cancer stem cells is not
characterized by overexpression by the cancer stem cell of one or
more Notch receptors, comprising administering to the human a
therapeutically effective amount of an antibody which binds to a
membrane proximal region of the extracellular domain of a Notch1
receptor and blocks ligand activation of a Notch1 receptor.
The invention further provides a method of treating cancer in a
human comprising administering to the human therapeutically
effective amounts of (a) a first antibody which binds a Notch1
receptor and inhibits growth of cancer stem cells which overexpress
Notch receptors; and (b) a second antibody which binds a Notch
receptor and blocks ligand activation of a Notch receptor.
The invention also provides another method of treating cancer,
wherein the cancer is selected from the group consisting of breast,
colon, pancreatic, prostate, lung, rectal and colorectal cancer,
comprising administering a therapeutically effective amount of an
antibody that blocks ligand activation of a Notch1 receptor.
The invention additionally provides: a humanized antibody which
binds Notch1 and blocks ligand activation of a Notch1 receptor; a
composition comprising the humanized antibody and a
pharmaceutically acceptable carrier; and an immunoconjugate
comprising the humanized antibody conjugated with a cytotoxic
agent.
Moreover, the invention provides an isolated polynucleotide
encoding the humanized antibody; a vector comprising the nucleic
acid; a host cell comprising the nucleic acid or the vector; as
well as a process of producing the humanized antibody comprising
culturing a host cell comprising the nucleic acid so that the
nucleic acid is expressed and, optionally, further comprising
recovering the humanized antibody from the host cell culture (e.g.,
from the host cell culture medium).
The invention further pertains to an immunoconjugate comprising an
antibody that binds Notch conjugated to one or more calicheamicin
molecules, and the use of such conjugates for treating Notch
expressing cancer, e.g., a cancer in which cancer stem cells
overexpress Notch.
Examples of solid tumors that can be treated using a therapeutic
composition of the instant invention, for example, an antibody that
binds a membrane promixal region of the extracellular domain of a
Notch1 receptor include, but are not limited to, sarcomas and
carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma.
Where aspects or embodiments of the invention are described in
terms of a Markush group or other grouping of alternatives, the
present invention encompasses not only the entire group listed as a
whole, but also each member of the group individually and all
possible subgroups of the main group, and also the main group
absent one or more of the group members. The present invention also
envisions the explicit exclusion of one or more of any of the group
members in the claimed invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Identification of Antibodies Targeting the Membrane
Proximal Region of Notch that Inhibit Notch Signaling.
(A) Schematic of the Notch receptor and 52M antigen region. The 52M
antigen includes the area of the Notch1 receptor subject to
cleavage by furin during maturation of the receptor and cleavage by
ADAM (A Disintegrin and Metalloprotease) proteases following ligand
binding. Subsequent processing by gamma-secretase causes the
release of the intracellular domain (ICD) of Notch that activates
gene transcription in the nucleus. (B) Luciferase levels (y-axis)
derived from Notch1-Hela cells cultured in the presence of a
soluble Notch ligand (hDLL4-fc) and Notch1 receptor antibodies.
Results from non-transfected (NT) cells with and without hDLL4-Fc
are shown on the far left of the x-axis. 52M Notch1 receptor
antibodies are shown along the x-axis and compared to DBZ, a Notch
gamma-secretase inhibitor (GSI), and 21M18, an anti-DLL4 antibody.
Notch1 receptor antibodies 52M51, 52M63, 52M74 and 52M80 all
significantly inhibited Notch signaling as indicated by a decrease
in luciferase activity. (C) Luciferase levels (y-axis) derived from
Notch1-Hela cells cultured in the presence of a soluble Notch
ligand (hDLL4-fc) and Notch1 receptor antibodies. Results from
non-transfected (NT) cells with and without hDLL4-Fc are shown on
the far left of the x-axis. 52M51 murine hybridoma derived antibody
and humanized variant 52M51-H4/L3 are shown along the x-axis in
various concentrations as indicated. Both the parental murine
antibody 52M51 and the humanized variant significantly inhibited
Notch signaling as indicated by a decrease in luciferase activity.
(D) Western blot analysis of ICD formation after ligand-mediated
stimulation of Notch1 expressing Hela cells. Minimal ICD is
produced in the absence of DLL4 ligand (-DLL4), but formation is
stimulated by the presence of DLL4. Antibodies 52M51, 52M63, 52M74,
and 52M80 reduce ICD formation to background levels despite the
presence of DLL4.
FIG. 2: Notch1 Receptor Antibody 52M51 Inhibits Tumor Formation In
vivo.
(A) NOD/SCID mice injected with C8 colon tumor cells were treated
with control antibody (squares) or anti-Notch1 antibody 52M51
(triangles), and tumor volume (y-axis, mm.sup.3) was measured
across time (x-axis, days). Treatment with 52M51 antibodies
significantly (p=0.0006) inhibited tumor growth compared to
control. (B) Individual tumor volume measurements from animals in
(A) measured on days 48 and 55 for control (left) versus 52M51
(right) treated mice. A line demarcates the average of each
experimental group. (C) NOD/SCID mice injected with PE13 breast
tumor cells were treated with control antibody (black squares) or
anti-Notch1 antibodies that do not inhibit Notch signaling as shown
in FIG. 1B: 52M1 (black triangles) and 52M2 (grey circles). Tumor
volume (y-axis, mm.sup.3) was measured across time (x-axis, days).
Treatment with 52M1 and 52M2 failed to effect tumor growth when
compared to control treated animals. (D) NOD/SCID mice injected
with PE13 breast tumor cells were treated with control antibody
(squares) or anti-Notch1 antibody 52M8 (triangles) that does not
inhibit Notch signaling as shown in FIG. 1B. Tumor volume (y-axis,
mm.sup.3) was measured across time (x-axis, days). Treatment with
52M8 failed to effect tumor growth when compared to control treated
animals.
FIG. 3: Anti-Notch1 Receptor Antibody 52R43 Inhibits Tumor Growth
In vivo
(A) NOD/SCID mice injected with M2 melanoma tumor cells were
treated with control antibody (squares) or anti-Notch1 antibody
52R43 (circles), and tumor volume (y-axis, mm.sup.3) was measured
across time (x-axis, days). (B) NOD/SCID mice injected with Lu24
lung tumor cells were treated with control antibody (squares) or
anti-Notch1 antibody 52R43 (circles), and tumor volume (y-axis,
mm.sup.3) was measured across time (x-axis, days). (C) NOD/SCID
mice injected with PN8 pancreatic tumor cells were treated with
control antibody (squares) or anti-Notch1 antibody 52R43 (circles),
and tumor volume (y-axis, mm.sup.3) was measured across time
(x-axis, days). (D) NOD/SCID mice injected with T1 breast tumor
cells were treated with control antibody (squares), anti-Notch1
antibody 52R43 (closed circles), taxol (triangles) or 52R43 and
taxol (open circles) and tumor volume (y-axis, mm.sup.3) was
measured across time (x-axis, days).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel agents, including, but not
limited to polypeptides such as antibodies, that bind one or more
human Notch receptors. The Notch-binding agents include antagonists
of the human Notch receptor(s). Related polypeptides and
polynucleotides, compositions comprising the Notch-binding agents,
and methods of making the Notch-binding agents are also provided.
Methods of using the novel Notch-binding agents, such as methods of
inhibiting tumor growth and/or treating cancer, are further
provided.
The present invention further identifies molecules (e.g.,
antibodies) that specifically bind to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor and inhibit tumor growth in vivo. The ligand binding
region of Notch, which is necessary and sufficient for ligand
binding, has been identified as EGF repeats 11 and 12, suggesting
this region of the Notch receptor is important in Notch signaling
and tumorigenesis (Rebay et al., 1991, Cell 67:687; Lei et al.,
2003, Dev. 130:6411; Hambleton et al., 2004, Structure 12:2173).
Unexpectedly, antibodies that bind outside the ligand binding
domain of the extracellular domain of human Notch receptor have
been found to inhibit tumor cell growth in vivo (see U.S. Patent
Publication No. 2008/0131434, incorporated by reference herein in
its entirety). Thus, antibodies that bind outside the ligand
binding domain of the extracellular domain of one or more of the
human Notch receptors--Notch1, Notch2, Notch3, and Notch4--have
value as potential cancer therapeutics.
Monoclonal antibodies that specifically bind to the membrane
proximal region of the extracellular domain of a Notch1, including
the monoclonal antibody 52M51, have now been identified (Example
1). Humanized 52M51 antibodies have also been generated (Example
2). Several of the antibodies, including 52M51 and a humanized
variant of 52M51, inhibit ligand-induced Notch1 signaling (Example
3 and FIGS. 1B and C), despite binding to Notch1 in a region
outside of the ligand-binding region. The ability of several of the
antibodies to inhibit formation of the Notch intracellular domain
(ICD) has also now been demonstrated (Example 3 and FIG. 1D). 52M51
has been found to inhibit tumor cell growth in vivo in a xenograft
model (Example 5 and FIGS. 2A and B). In addition, another antibody
52R43 has been found to inhibit tumor cell growth in vivo in
multiple xenograft models (Example 7 and FIG. 3A-D).
Definitions
An "antagonist" of a Notch receptor as used herein is a term that
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of the Notch pathway. Suitable
antagonist molecules specifically include antagonist antibodies or
antibody fragments. The term "antagonist" is used herein to include
any molecule that partially or fully blocks, inhibits, or
neutralizes the expression of a Notch receptor.
The term "antibody" is used to mean an immunoglobulin molecule that
recognizes and specifically binds to a target, such as a protein,
polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing etc., through at least one antigen
recognition site within the variable region of the immunoglobulin
molecule. As used herein, the term encompasses intact polyclonal
antibodies, intact monoclonal antibodies, antibody fragments (such
as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv)
mutants, multispecific antibodies such as bispecific antibodies
generated from at least two intact antibodies, monovalent or
monospecific antibodies, chimeric antibodies, humanized antibodies,
human antibodies, fusion proteins comprising an antigen
determination portion of an antibody, and any other modified
immunoglobulin molecule comprising an antigen recognition site so
long as the antibodies exhibit the desired biological activity. An
antibody can be any of the five major classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the
identity of their heavy-chain constant domains referred to as
alpha, delta, epsilon, gamma, and mu, respectively. The different
classes of immunoglobulins have different and well known subunit
structures and three-dimensional configurations. Antibodies can be
naked or conjugated to other molecules such as toxins,
radioisotopes, etc.
As used herein, the term "antibody fragment" refers to a portion of
an intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to Fab, Fab', F(ab')2, and Fv
fragments, linear antibodies, single chain antibodies, and
multispecific antibodies formed from antibody fragments.
An "Fv antibody" refers to the minimal antibody fragment that
contains a complete antigen-recognition and -binding site either as
two-chains, in which one heavy and one light chain variable domain
form a non-covalent dimer, or as a single-chain (scFv), in which
one heavy and one light chain variable domain are covalently linked
by a flexible peptide linker so that the two chains associate in a
similar dimeric structure. In this configuration the complementary
determining regions (CDRs) of each variable domain interact to
define the antigen-binding specificity of the Fv dimer.
Alternatively a single variable domain (or half of an Fv) can be
used to recognize and bind antigen, although generally with lower
affinity.
A "monoclonal antibody" as used herein refers to homogenous
antibody population involved in the highly specific recognition and
binding of a single antigenic determinant, or epitope. This is in
contrast to polyclonal antibodies that typically include different
antibodies directed against different antigenic determinants. The
term "monoclonal antibody" encompasses both intact and full-length
monoclonal antibodies as well as antibody fragments (e.g., Fab,
Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins
comprising an antibody portion, and any other modified
immunoglobulin molecule comprising an antigen recognition site.
Furthermore, "monoclonal antibody" refers to such antibodies made
in any number of manners including but not limited to by hybridoma,
phage selection, recombinant expression, and transgenic
animals.
As used herein, the term "humanized antibody" refers to forms of
non-human (e.g., murine) antibodies that are specific
immunoglobulin chains, chimeric immunoglobulins, or fragments
thereof that contain minimal non-human sequences. Typically,
humanized antibodies are human immunoglobulins in which residues
from the complementary determining regions (CDRs) are replaced by
residues from a CDR of a non-human species (e.g., mouse., rat,
rabbit, hamster, etc.) that have the desired specificity, affinity,
and/or capability. In some instances, the Fv framework region (FR)
residues of a human immunoglobulin are replaced with the
corresponding residues in an antibody from a non-human species that
has the desired specificity, affinity, and/or capability. The
humanized antibody can be further modified by the substitution of
additional residues either in the Fv framework region and/or within
the replaced non-human residues to refine and optimize antibody
specificity, affinity, and/or capability. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two or three, variable domains containing all, or
substantially all, of the CDR regions that correspond to the
non-human immunoglobulin whereas all, or substantially all, of the
FR regions are those of a human immunoglobulin consensus sequence.
The humanized antibody can also comprise at least a portion of an
immunoglobulin constant region or domain (Fc), typically that of a
human immunoglobulin. Examples of methods used to generate
humanized antibodies are described in U.S. Pat. No. 5,225,539,
herein incorporated by reference.
A "variable region" of an antibody refers to the variable region of
the antibody light chain or the variable region of the antibody
heavy chain, either alone or in combination. The variable regions
of the heavy and light chain each consist of four framework regions
(FR) connected by three complementarity determining regions (CDRs)
also known as hypervariable regions. The CDRs in each chain are
held together in close proximity by the FRs and, with the CDRs from
the other chain, contribute to the formation of the antigen-binding
site of antibodies. There are at least two techniques for
determining CDRs: (1) an approach based on cross-species sequence
variability (i.e., Kabat et al. Sequences of Proteins of
Immunological Interest, 5th ed., 1991, National Institutes of
Health, Bethesda, Md.); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (Al-lazikani
et al., 1997, J. Molec. Biol. 273:927-948). In addition,
combinations of these two approaches are sometimes used in the art
to determine CDRs.
The term "human antibody" as used herein means an antibody produced
by a human or an antibody having an amino acid sequence
corresponding to an antibody produced by a human made using any of
the techniques known in the art. This definition of a human
antibody includes intact or full-length antibodies, fragments
thereof, and/or antibodies comprising at least one human heavy
and/or light chain polypeptide such as, for example, an antibody
comprising murine light chain and human heavy chain
polypeptides.
The term "chimeric antibodies" refers to antibodies wherein the
amino acid sequence of the immunoglobulin molecule is derived from
two or more species. Typically, the variable region of both light
and heavy chains corresponds to the variable region of antibodies
derived from one species of mammals (e.g., mouse, rat, rabbit,
etc.) with the desired specificity, affinity, and/or capability,
while the constant regions are homologous to the sequences in
antibodies derived from another species (usually human) to avoid
eliciting an immune response in that species. The term chimeric
antibody includes monovalent, divalent and polyvalent
antibodies.
The term "epitope" or "antigenic determinant" are used
interchangeably herein and refer to that portion of an antigen
capable of being recognized and specifically bound by a particular
antibody. When the antigen is a polypeptide, epitopes can be formed
both from contiguous amino acids and noncontiguous amino acids
juxtaposed by tertiary folding of a protein. Epitopes formed from
contiguous amino acids (also referred to as linear epitopes) are
typically retained upon protein denaturing, whereas epitopes formed
by tertiary folding (also referred to as conformational epitopes)
are typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation.
That an antibody "selectively binds" or "specifically binds" to an
epitope or receptor means that the antibody reacts or associates
more frequently, more rapidly, with greater duration, with greater
affinity, or with some combination of the above to the epitope or
receptor than with alternative substances, including unrelated
proteins. "Selectively binds" or "specifically binds" means, for
instance, that an antibody binds to a protein with a K.sub.D of
about 0.1 mM or less, at times about 1 .mu.M or less, at times
about 0.1 .mu.M or less and at times about 0.01 .mu.M or less.
Because of the sequence identity between homologous proteins in
different species, specific binding can include an antibody that
recognizes a Notch receptor in more than one species. It is
understood that, in certain embodiments, an antibody or binding
moiety that specifically binds to a first target may or may not
specifically bind to a second target. As such, "specific binding"
does not necessarily require (although it can include) exclusive
binding, i.e. binding to a single target. Generally, but not
necessarily, reference to binding means specific binding.
Competition between antibodies is determined by an assay in which
the immunoglobulin under study inhibits specific binding of a
reference antibody to a common antigen. Numerous types of
competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phase
direct biotin-avidin EIA (see Kirkland et al., J. Immmunol.
137:3614-3619 (1986)); solid phase direct labeled assay, solid
phase direct labeled sandwich assay (see Harlow and Lane,
"Antibodies, A Laboratory Manual," Cold Spring Harbor Press
(1988)); solid phase direct label RIA using .sup.125I label (see
Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase
direct biotin-avidin EIA (Cheung et al., Virology 176:546-552
(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J.
Immunol. 32:77-82 (1990)). Typically, such an assay involves the
use of purified antigen bound to a solid surface or cells bearing
either of these, an unlabeled test immunoglobulin and a labeled
reference immunoglobulin. Competitive inhibition is measured by
determining the amount of label bound to the solid surface or cells
in the presence of the test immunoglobulin. Usually the test
immunoglobulin is present in excess. Antibodies identified by
competition assay (competing antibodies) include antibodies binding
to the same epitope as the reference antibody and antibodies
binding to an adjacent epitope sufficiently proximal to the epitope
bound by the reference antibody for steric hindrance to occur.
Usually, when a competing antibody is present in excess, it will
inhibit specific binding of a reference antibody to a common
antigen by at least 50 or 75%.
The terms "isolated" or "purified" refer to material that is
substantially or essentially free from components that normally
accompany it in its native state. Purity and homogeneity are
typically determined using analytical chemistry techniques such as
polyacrylamide gel electrophoresis or high performance liquid
chromatography. A protein (e.g., an antibody) or nucleic acid that
is the predominant species present in a preparation is
substantially purified. In particular, in some embodiments, an
isolated nucleic acid comprising a gene is separated from open
reading frames that naturally flank the gene and encode proteins
other than the protein encoded by the gene. An isolated antibody is
separated from other non-immunoglobulin proteins and from other
immunoglobulin proteins with different antigen binding
specificities. It can also mean that the nucleic acid or protein is
at least 85% pure, at least 95% pure, and in some embodiments, at
least 99% pure.
As used herein, the terms "cancer" and "cancerous" refer to or
describe the physiological condition in mammals in which a
population of cells are characterized by unregulated cell growth.
Examples of cancer include, but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia. More particular examples
of such cancers include, but are not limited to, squamous cell
cancer, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, squamous carcinoma of the lung, cancer
of the peritoneum, hepatocellular cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, liver cancer, prostate
cancer, vulval cancer, thyroid cancer, hepatic carcinoma and
various types of head and neck cancer.
"Tumor" and "neoplasm" as used herein refer to any mass of tissue
that result from excessive cell growth or proliferation, either
benign (noncancerous) or malignant (cancerous) including
pre-cancerous lesions.
The terms "proliferative disorder" and "proliferative disease"
refer to disorders associated with abnormal cell proliferation such
as cancer.
"Metastasis" as used herein refers to the process by which a cancer
spreads or transfers from the site of origin to other regions of
the body with the development of a similar cancerous lesion at the
new location. A "metastatic" or "metastasizing" cell is one that
loses adhesive contacts with neighboring cells and migrates via the
bloodstream or lymph from the primary site of disease to invade
neighboring body structures.
The terms "cancer stem cell" or "tumor stem cell" or "solid tumor
stem cell" are used interchangeably herein and refer to a
population of cells from a solid tumor that: (1) have extensive
proliferative capacity; 2) are capable of asymmetric cell division
to generate one or more kinds of differentiated progeny with
reduced proliferative or developmental potential; and (3) are
capable of symmetric cell divisions for self-renewal or
self-maintenance. These properties of "cancer stem cells" or "tumor
stem cells" or "solid tumor stem cells" confer on those cancer stem
cells the ability to form palpable tumors upon serial
transplantation into an immunocompromised mouse compared to the
majority of tumor cells that fail to form tumors. Cancer stem cells
undergo self-renewal versus differentiation in a chaotic manner to
form tumors with abnormal cell types that can change over time as
mutations occur.
The terms "cancer cell" or "tumor cell" refer to the total
population of cells derived from a tumor including both
non-tumorigenic cells, which comprise the bulk of the tumor cell
population, and tumorigenic stem cells (cancer stem cells).
As used herein "tumorigenic" refers to the functional features of a
solid tumor stem cell including the properties of self-renewal
(giving rise to additional tumorigenic cancer stem cells) and
proliferation to generate all other tumor cells (giving rise to
differentiated and thus non-tumorigenic tumor cells) that allow
solid tumor stem cells to form a tumor.
As used herein, the "tumorigenicity" of a tumor refers to the
ability of a random sample of cells from the tumor to form palpable
tumors upon serial transplantation into immunocompromised mice.
As used herein, the terms "stem cell cancer marker" or "cancer stem
cell marker" or "tumor stem cell marker" or "solid tumor stem cell
marker" refer to a gene or genes or a protein, polypeptide, or
peptide expressed by the gene or genes whose expression level,
alone or in combination with other genes, is correlated with the
presence of tumorigenic cancer cells compared to non-tumorigenic
cells. The correlation can relate to either an increased or
decreased expression of the gene (e.g., increased or decreased
levels of mRNA or the peptide encoded by the gene).
The terms "cancer stem cell gene signature" or "tumor stem cell
gene signature" or "cancer stem cell signature" are used
interchangeably herein to refer to gene signatures comprising genes
differentially expressed in cancer stem cells compared to other
cells or population of cells, for example normal breast epithelial
tissue. In some embodiments the cancer stem cell gene signatures
comprise genes differentially expressed in cancer stem cells versus
normal breast epithelium by a fold change, for example by 2 fold
reduced and/or elevated expression, and further limited by using a
statistical analysis such as, for example, by the P value of a
t-test across multiple samples. In another embodiment, the genes
differentially expressed in cancer stem cells are divided into
cancer stem cell gene signatures based on the correlation of their
expression with a chosen gene in combination with their fold or
percentage expression change. Cancer stem cell signatures are
predictive both retrospectively and prospectively of an aspect of
clinical variability, including but not limited to, metastasis and
death.
The term "genetic test" as used herein refers to procedures whereby
the genetic make-up of a patient or a patient tumor sample is
analyzed. The analysis can include detection of DNA, RNA,
chromosomes, proteins or metabolites to detect heritable or somatic
disease-related genotypes or karyotypes for clinical purposes.
As used herein, the terms "biopsy" or "biopsy tissue" refer to a
sample of tissue or fluid that is removed from a subject for the
purpose of determining if the sample contains cancerous tissue. In
some embodiments, biopsy tissue or fluid is obtained because a
subject is suspected of having cancer. The biopsy tissue or fluid
is then examined for the presence or absence of cancer.
As used herein, the term "subject" refers to any animal (e.g., a
mammal), including, but not limited to humans, non-human primates,
rodents, and the like, which is to be the recipient of a particular
treatment. Typically, the terms "subject" and "patient" are used
interchangeably herein in reference to a human subject.
"Pharmaceutically acceptable" refers to approved or approvable by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, including humans.
"Pharmaceutically acceptable excipient, carrier or adjuvant" or
"acceptable pharmaceutical carrier" refers to an excipient, carrier
or adjuvant that can be administered to a subject, together with at
least one antibody of the present disclosure, and which does not
destroy the pharmacological activity thereof and is nontoxic when
administered in doses sufficient to deliver a therapeutic amount of
the antibody. In addition, a "pharmaceutically acceptable carrier"
does not trigger an immune response in a recipient subject.
Examples include, but are not limited to, any of the standard
pharmaceutical carriers such as a phosphate buffered saline
solution, water, and various oil/water emulsions. Some diluents for
aerosol or parenteral administration are phosphate buffered saline
or normal (0.9%) saline.
"Pharmaceutically acceptable vehicle" refers to a diluents,
adjuvant, excipient, or carrier with which at least one antibody of
the present disclosure is administered.
The term "effective amount" or "therapeutically effective amount"
or "therapeutic effect" refers to an amount of an antibody,
polypeptide, polynucleotide, small organic molecule, or other drug
effective to "treat" a disease or disorder in a subject or mammal.
In the case of cancer, the therapeutically effective amount of the
drug has a therapeutic effect and as such can reduce the number of
cancer cells; decrease tumorigenicity, tumorigenic frequency or
tumorigenic capacity; reduce the number or frequency of cancer stem
cells; reduce the tumor size; inhibit or stop cancer cell
infiltration into peripheral organs including, for example, the
spread of cancer into soft tissue and bone; inhibit and stop tumor
metastasis; inhibit and stop tumor growth; relieve to some extent
one or more of the symptoms associated with the cancer; reduce
morbidity and mortality; improve quality of life; or a combination
of such effects. To the extent the agent, for example an antibody,
prevents growth and/or kills existing cancer cells, it can be
referred to as cytostatic and/or cytotoxic.
Terms such as "treating" or "treatment" or "to treat" or
"alleviating" or "to alleviate" refer to both 1) therapeutic
measures that cure, slow down, lessen symptoms of, and/or halt
progression of a diagnosed pathologic condition or disorder and 2)
prophylactic or preventative measures that prevent or slow the
development of a targeted pathologic condition or disorder. Thus
those in need of treatment include those already with the disorder;
those prone to have the disorder; and those in whom the disorder is
to be prevented. A subject is successfully "treated" according to
the methods of the present invention if the patient shows one or
more of the following: a reduction in the number of or complete
absence of cancer cells; a reduction in the tumor size; inhibition
of or an absence of cancer cell infiltration into peripheral organs
including the spread of cancer into soft tissue and bone;
inhibition of or an absence of tumor metastasis; inhibition or an
absence of tumor growth; relief of one or more symptoms associated
with the specific cancer; reduced morbidity and mortality;
improvement in quality of life; reduction in tumorigenicity;
reduction in the number or frequency of cancer stem cells; or some
combination of effects.
As used herein, the terms "polynucleotide" or "nucleic acid" refer
to a polymer composed of a multiplicity of nucleotide units
(ribonucleotide or deoxyribonucleotide or related structural
variants) linked via phosphodiester bonds, including but not
limited to, DNA or RNA. The term encompasses sequences that include
any of the known base analogs of DNA and RNA.
The term "gene" refers to a nucleic acid (e.g., DNA) sequence that
comprises coding sequences necessary for the production of a
polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide
can be encoded by a full length coding sequence or by any portion
of the coding sequence so long as the desired activities or
functional properties (e.g., enzymatic activity, ligand binding,
signal transduction, immunogenicity, etc.) of the full-length
polypeptide or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb or more on either end such that the gene
corresponds to the length of the full-length mRNA. The term "gene"
encompasses both cDNA and genomic forms of a gene.
The term "recombinant" when used with reference to a cell, nucleic
acid, protein or vector indicates that the cell, nucleic acid,
protein or vector has been modified by the introduction of a
heterologous nucleic acid or protein, the alteration of a native
nucleic acid or protein, or that the cell is derived from a cell so
modified. Thus, e.g., recombinant cells express genes that are not
found within the native (non-recombinant) form of the cell or
express native genes that are overexpressed or otherwise abnormally
expressed such as, for example, expressed as non-naturally
occurring fragments or splice variants. By the term "recombinant
nucleic acid" herein is meant nucleic acid, originally formed in
vitro, in general, by the manipulation of nucleic acid, e.g., using
polymerases and endonucleases, in a form not normally found in
nature. In this manner, operably linkage of different sequences is
achieved. Thus an isolated nucleic acid, in a linear form, or an
expression vector formed in vitro by ligating DNA molecules that
are not normally joined, are both considered recombinant for the
purposes of this invention. It is understood that once a
recombinant nucleic acid is made and introduced into a host cell or
organism, it will replicate non-recombinantly, i.e., using the in
vivo cellular machinery of the host cell rather than in vitro
manipulations; however, such nucleic acids, once produced
recombinantly, although subsequently replicated non-recombinantly,
are still considered recombinant for the purposes of the invention.
Similarly, a "recombinant protein" is a protein made using
recombinant techniques, i.e., through the expression of a
recombinant nucleic acid as depicted above.
As used herein, the term "vector" is used in reference to nucleic
acid molecules that transfer DNA segment(s) from one cell to
another. Vectors are often derived from plasmids, bacteriophages,
or plant or animal viruses.
As used herein, the term "gene expression" refers to the process of
converting genetic information encoded in a gene into RNA (e.g.,
mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene
(e.g., via the enzymatic action of an RNA polymerase), and for
protein encoding genes, into protein through "translation" of mRNA.
Gene expression can be regulated at many stages in the process.
"Up-regulation" or "activation" refers to regulation that increases
the production of gene expression products (e.g., RNA or protein),
while "down-regulation" or "repression" refers to regulation that
decrease production. Molecules (e.g., transcription factors) that
are involved in up-regulation or down-regulation are often called
"activators" and "repressors," respectively.
The terms "polypeptide" or "peptide" or "protein" or "protein
fragment" are used interchangeably herein to refer to a polymer of
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residue is an artificial chemical
mimetic of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers and non-naturally
occurring amino acid polymers.
The term "amino acid" refers to naturally occurring and synthetic
amino acids, as well as amino acid analogs and amino acid mimetics
that function similarly to the naturally occurring amino acids.
Naturally occurring amino acids are those encoded by the genetic
code, as well as those amino acids that are later modified, e.g.,
hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. "Amino
acid analogs" refers to compounds that have the same basic chemical
structure as a naturally occurring amino acid, e.g., an alpha
carbon that is bound to a hydrogen, a carboxyl group, an amino
group, and an R group, e.g., homoserine, norleucine, methionine
sulfoxide, methionine methyl sulfonium. Such analogs can have
modified R groups (e.g., norleucine) or modified peptide backbones,
but retain the same basic chemical structure as a naturally
occurring amino acid. "Amino acid mimetics" refers to chemical
compounds that have a structure that is different from the general
chemical structure of an amino acid, but that functions similarly
to a naturally occurring amino acid.
"Conservatively modified variants" applies to both amino acid and
nucleic acid sequences. "Amino acid variants" refers to amino acid
sequences. With respect to particular nucleic acid sequences,
conservatively modified variants refers to those nucleic acids
which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical or associated (e.g., naturally
contiguous) sequences. Because of the degeneracy of the genetic
code, a large number of functionally identical nucleic acids encode
most proteins. For instance, the codons GCA, GCC, GCG and GCU all
encode the amino acid alanine. Thus, at every position where an
alanine is specified by a codon, the codon can be altered to
another of the corresponding codons described without altering the
encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
It is recognized that in certain contexts each codon in a nucleic
acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, silent variations of a nucleic acid which
encodes a polypeptide is implicit in a described sequence with
respect to the expression product, but not with respect to actual
probe sequences.
As to amino acid sequences, it will be recognized that individual
substitutions, deletions or additions to a nucleic acid, peptide,
polypeptide, or protein sequence which alters, adds or deletes a
single amino acid or a small percentage of amino acids in the
encoded sequence is a "conservatively modified variant" including
where the alteration results in the substitution of an amino acid
with a chemically similar amino acid. Conservative substitution
tables providing functionally similar amino acids are well known in
the art. (See, for example, Table 1). Guidance concerning which
amino acid changes are likely to be phenotypically silent can also
be found in Bowie et al., 1990, Science 247:1306 1310. Such
conservatively modified variants are in addition to and do not
exclude polymorphic variants, interspecies homologs, and alleles of
the invention. Typically conservative substitutions include: 1)
Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,
Creighton, Proteins (1984)). As indicated, changes are typically of
a minor nature, such as conservative amino acid substitutions that
do not significantly affect the folding or activity of the
protein.
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions
Original Amino Acid Exemplary Conservative Substitutions Alanine
Valine, Isoleucine, Leucine, Glycine, Serine Arginine Lysine,
Histidine, Glutamine, Asparagine Asparagine Glutamine, Histidine,
Lysine, Arginine Aspartic Acid Glutamic Acid, Asparagine Cysteine
Serine, Alanine, Methionine Glutamine Asparagine Glutamic Acid
Aspartic Acid, Glutamine Glycine Proline, Alanine Histidine
Asparagine, Glutamine, Lysine, Arginine Isoleucine Leucine, Valine,
Methionine, Alanine, Phenylalanine, Norleucine Leucine Norleucine,
Isoleucine, Valine, Methionine, Alanine, Phenylalanine Lysine
Arginine, Glutamine, Asparagine, Histidine Methionine Leucine,
Phenylalanine, Isoleucine, Valine, Cysteine Phenylalanine Leucine,
Valine, Isoleucine, Alanine, Tyrosine Proline Alanine, Glycine
Serine Threonine Threonine Serine Trytophan Tyrosine, Phenylalanine
Tyrosine Tryptophan, Phenylalanine, Threonine, Serine Valine
Isoleucine, Methionine, Leucine, Phenylalanine, Alanine,
Norleucine
As used in the present disclosure and claims, the singular forms
"a", "an", and "the" include plural forms unless the context
clearly dictates otherwise.
It is understood that whenever embodiments are described herein
with the language "comprising" otherwise analogous embodiments
described in terms of "consisting" and/or "consisting essentially
of" are also provided.
Certain Embodiments of the Present Invention
The present invention provides compositions and methods for
studying, diagnosing, characterizing, and treating cancer. In
particular, in certain embodiments, the present invention provides
agents, including antagonists, that bind Notch receptors and
methods of using the agents or antagonists to inhibit tumor growth
and treat cancer or other diseases in human patients. In certain
embodiments, the antagonists are antibodies that specifically bind
to a non-ligand binding region of the extracellular domain of a
human Notch receptor.
In one aspect, the present invention provides an antibody that
specifically binds to a non-ligand binding membrane proximal region
of the extracellular domain of a human Notch1 receptor. In some
embodiments, the antibody binds a region of human Notch1 comprising
about amino acid 1427 to about amino acid 1732. In some
embodiments, the antibody binds to a region comprising SEQ ID NO:2.
In certain embodiments, the antibody that specifically binds to a
non-ligand binding membrane proximal region of the extracellular
domain of at least one additional Notch receptor.
In some embodiments, the antibody is an antagonist of human Notch1.
In certain embodiments, the antibody inhibits ligand-induced
signaling of a Notch1 pathway. In some embodiments, the antibody
inhibits the activity of Notch1. In other embodiments the antibody
inhibits cleavage of a Notch1 receptor. In some embodiments, the
antibody inhibits cleavage of Notch1 at a site within the membrane
proximal region of the extracellular domain. In certain
embodiments, the antibody inhibits release or formation of the
intracellular domain (ICD) of Notch1. In other embodiments, the
antibody reduces the tumorigenicity of a tumor that comprises
cancer stem cells. In certain embodiments, the antibody inhibits
the growth of a tumor comprising cancer stem cells. In certain
embodiments, the antibody inhibits the growth of a tumor.
In certain embodiments, the antibody that specifically binds to a
membrane proximal region of the extracellular domain of a human
Notch1 receptor and inhibits tumor growth is a monoclonal antibody.
In certain embodiments, the antibody that specifically binds to a
membrane proximal region of the extracellular domain of a human
Notch1 receptor is a chimeric antibody, is a humanized antibody, is
a human antibody, is an antibody fragment, or is a bispecific
antibody. In certain embodiments, the present invention provides a
hybridoma producing an antibody that specifically binds to a
non-ligand binding membrane proximal region of the extracellular
domain of a human Notch1 receptor and inhibits tumor growth.
In another aspect, the invention provides a method of inhibiting
the growth of a tumor in a subject, the method comprising
administering to the subject a therapeutically effective amount of
an antibody that specifically binds to a non-ligand binding
membrane proximal region of the extracellular domain of a human
Notch1 receptor protein. In some embodiments, the tumor comprises
cancer stem cells. In some embodiments, the methods comprise
targeting the cancer stem cells with the antibodies. In certain
embodiments, the method of inhibiting growth of a tumor comprises
administering a therapeutically effective amount of a monoclonal
antibody. In certain embodiments, the method of inhibiting growth
of a tumor comprises administering a therapeutically effective
amount of a chimeric antibody. In certain embodiments, the method
of inhibiting growth of a tumor comprises administering a
therapeutically effective amount of a humanized antibody. In
certain embodiments, the method of inhibiting growth of a tumor
comprises administering a therapeutically effective amount of a
human antibody.
In certain embodiments, the method of inhibiting growth of a tumor
comprises reducing the frequency of cancer stem cells in the tumor,
reducing the number of cancer stem cells in the tumor, reducing the
tumorigenicity of the tumor, and/or reducing the tumorigenicity of
the tumor by reducing the number or frequency of cancer stem cells
in the tumor. In some embodiments, the method of inhibiting growth
of a tumor comprises inhibiting the activity of a Notch1 receptor.
In certain embodiments, the tumor includes, but is not limited to,
a breast tumor, colorectal tumor, hepatic tumor, renal tumor, lung
tumor, pancreatic tumor, ovarian tumor, prostate tumor and head and
neck tumor.
In another aspect, the present invention provides a method of
treating cancer in a subject in need thereof comprising
administering to a subject a therapeutically effective amount of an
antibody that specifically binds to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor protein and inhibits tumor growth in the subject. In
certain embodiments, the method of treating cancer comprises
administering a therapeutically effective amount of a monoclonal
antibody. In certain embodiments, the method of treating cancer
comprises administering a therapeutically effective amount of a
chimeric antibody. In certain embodiments, the method of treating
cancer comprises administering a therapeutically effective amount
of a humanized antibody. In certain embodiments, the method of
treating cancer comprises administering a therapeutically effective
amount of a human antibody.
In certain embodiments, the method of treating cancer comprises
administering a therapeutically effective amount of an antibody
conjugated to a cytotoxic moiety that specifically binds to a
non-ligand binding membrane proximal region of the extracellular
domain of a human Notch1 receptor and inhibits tumor growth. In
certain embodiments, the method of treating cancer comprises
administering a therapeutically effective amount of an antibody of
any of the aspects and/or embodiments, as well as other aspects
and/or embodiments described herein, in combination with radiation
therapy. In certain embodiments, the method of treating cancer
comprises administering a therapeutically effective amount of an
antibody of any of the aspects and/or embodiments, as well as other
aspects and/or embodiments described herein, in combination with
chemotherapy. In certain embodiments, the method of treating cancer
comprises administering a therapeutically effective amount of an
antibody that specifically binds to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor and inhibits tumor growth that are from tumors including,
but not limited to, a breast tumor, colorectal tumor, lung tumor,
pancreatic tumor, prostate tumor, or a head and neck tumor.
In certain embodiments, the method of treating cancer comprises
identifying patients in need of treatment using a genetic test
comprising an antibody that specifically binds to a non-ligand
binding membrane proximal region of the extracellular domain of a
human Notch1 receptor; and administering a therapeutically
effective amount of the antibody to the patients. In certain
embodiments, the method of treating cancer comprises identifying
patients in need of treatment with an antibody that specifically
binds to a non-ligand binding membrane proximal region of the
extracellular domain of a human Notch1 receptor using a genetic
test that detects a cancer stem cell signature, and administering a
therapeutically effective amount of the antibody that specifically
binds to a non-ligand binding membrane proximal region of the
extracellular domain of a human Notch1 receptor and inhibits tumor
growth.
In another aspect, the present invention provides a method of
identifying a molecule that binds to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor and inhibits tumor growth, the method comprising: i)
incubating the molecule with a non-ligand binding membrane proximal
region of the extracellular domain of a human Notch1 receptor; ii)
determining if the molecule binds to the non-ligand binding
membrane proximal region of the extracellular domain of the human
Notch1 receptor; and iii) determining if the molecule inhibits
tumor growth. In certain embodiments, the invention provides a
method of identifying a molecule that binds to a non-ligand binding
membrane proximal region of an extracellular domain of a human
Notch1 receptor and inhibits tumor growth, the method comprising:
i) incubating the molecule with the non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor comprising SEQ ID NO:2; ii) determining if the molecule
binds to the non-ligand binding membrane proximal region of the
extracellular domain of the human Notch1 receptor comprising SEQ ID
NO:2; and iii) determining if the molecule inhibits tumor
growth.
In certain embodiments, the present invention provides a
pharmaceutical composition comprising an antibody that specifically
binds to a non-ligand binding membrane proximal region of the
extracellular domain of a human Notch1 receptor and inhibits tumor
growth.
In certain embodiments, the present invention provides a method of
making an antibody that specifically binds to a non-ligand binding
membrane proximal region of the extracellular domain of a human
Notch1 receptor and inhibits tumor growth.
In certain embodiments, the present invention provides an isolated
nucleic acid that encodes an antibody that specifically binds to a
non-ligand membrane proximal binding region of the extracellular
domain of a human Notch1 receptor and inhibits tumor growth.
In certain embodiments, antagonists against a Notch receptor, such
as Notch1, act extracellularly to act upon or inhibit the function
of the Notch receptor. In certain embodiments, an antagonist of a
Notch receptor is proteinaceous. In some embodiments, proteinaceous
antagonists of a Notch1 receptor are antibodies that specifically
bind to an extracellular epitope of a Notch1 receptor.
Extracellular binding of an antagonist against a Notch1 receptor
can inhibit the signaling of a Notch receptor by inhibiting
intrinsic activation (e.g. kinase activity) of a Notch1 receptor
and/or by sterically inhibiting the interaction, for example, of a
Notch receptor with one of its ligands. Furthermore, extracellular
binding of an antagonist to a Notch receptor can downregulate
cell-surface expression of a Notch receptor such as, for example,
by internalization of a Notch receptor and/or decreasing cell
surface trafficking of a Notch receptor. Extracellular binding of
an antagonist to a Notch receptor can inhibit cleavage of the Notch
receptor and reduce release of the LCD of Notch.
In some embodiments, antagonists against a Notch receptor bind to a
Notch receptor and have one or more of the following effects:
inhibit proliferation of tumor cells, trigger cell death directly
in tumor cells, or prevent metastasis of tumor cells. In certain
embodiments, antagonists of a Notch receptor trigger cell death via
a conjugated toxin, chemotherapeutic agent, radioisotope, or other
such agent. For example, an antibody against a Notch receptor is
conjugated to a toxin that is activated in tumor cells expressing
the Notch receptor by protein internalization. In other
embodiments, antagonists of a Notch receptor mediate cell death of
a cell expressing the Notch receptor via antibody-dependent
cellular cytotoxicity (ADCC). ADCC involves cell lysis by effector
cells that recognize the Fc portion of an antibody. Many
lymphocytes, monocytes, tissue macrophages, granulocytes and
eosinophils, for example, have Fc receptors and can mediate
cytolysis (Dillman, 1994, J. Clin. Oncol. 12:1497). In some
embodiments, an antagonist of a Notch receptor is an antibody that
triggers cell death of cell expressing a Notch receptor by
activating complement-dependent cytotoxicity (CDC). CDC involves
binding of serum complement to the Fc portion of an antibody and
subsequent activation of the complement protein cascade, resulting
in cell membrane damage and eventual cell death. Biological
activity of antibodies is known to be determined, to a large
extent, by the constant domains or Fc region of the antibody
molecule (Uananue and Benacerraf, Textbook of Immunology, 2nd
Edition, Williams & Wilkins, p. 218 (1984)). Antibodies of
different classes and subclasses differ in this respect, as do
antibodies of the same subclass but from different species. Of
human antibodies, IgM is the most efficient class of antibodies to
bind complement, followed by IgG1, IgG3, and IgG2 whereas IgG4
appears quite deficient in activating the complement cascade
(Dillman, 1994, J. Clin. Oncol. 12:1497; Jefferis et al., 1998,
Immunol. Rev. 163:59-76). According to the present invention,
antibodies of those classes having the desired biological activity
are prepared.
The ability of any particular antibody against a Notch receptor to
mediate lysis of the target cell by complement activation and/or
ADCC can be assayed. The cells of interest are grown and labeled in
vitro; the antibody is added to the cell culture in combination
with either serum complement or immune cells which can be activated
by the antigen antibody complexes. Cytolysis of the target cells is
detected, for example, by the release of label from the lysed
cells. In fact, antibodies can be screened using the patient's own
serum as a source of complement and/or immune cells. The antibody
that is capable of activating complement or mediating ADCC in the
in vitro test can then be used therapeutically in that particular
patient.
In certain embodiments, the Notch-binding agent or antagonist is an
antibody that does not have one or more effector functions. For
instance, in some embodiments, the antibody has no
antibody-dependent cellular cytotoxicity (ADCC) activity, and/or no
complement-dependent cytoxicity (CDC) activity. In certain
embodiments, the antibody does not bind to the Fc receptor and/or
complement factors. In certain embodiments, the antibody has no
effector function.
In other embodiments, antagonists of a Notch receptor can trigger
cell death indirectly by inhibiting angiogenesis. Angiogenesis is
the process by which new blood vessels form from pre-existing
vessels and is a fundamental process required for normal growth,
for example, during embryonic development, wound healing and in
response to ovulation. Solid tumor growth larger than 1-2 mm.sup.2
also requires angiogenesis to supply nutrients and oxygen without
which tumor cells die. Thus in certain embodiments, an antagonist
of a Notch receptor targets vascular cells that express the Notch
receptor including, for example, endothelial cells, smooth muscle
cells or components of the extracellular matrix required for
vascular assembly. In other embodiments, an antagonist of a Notch
receptor inhibits growth factor signaling required by vascular cell
recruitment, assembly, maintenance or survival.
The present invention provides a variety of polypeptides, including
but not limited to antibodies and fragments of antibodies. In
certain embodiments, the polypeptide is isolated. In certain
alternative embodiments, the polypeptide is substantially pure.
In certain embodiments, the polypeptides of the present invention
can be recombinant polypeptides, natural polypeptides, or synthetic
polypeptides comprising the sequence of SEQ ID NO:8, SEQ ID NO:14,
SEQ ID NO:24, SEQ ID NO:28, or SEQ ID NO:32 (with or without the
indicated signal sequences).
The invention provides a polypeptide comprising the heavy chain
and/or the light chain provided in SEQ ID NO:10 and/or SEQ ID NO:4,
respectively (with or without the indicated putative signal
sequences). In certain embodiments, the polypeptide is an antibody.
In certain embodiments, the polypeptide specifically binds a
non-ligand binding membrane proximal region of the extracellular
domain of a human Notch1 receptor.
The invention further provides a polypeptide comprising SEQ ID
NO:8, SEQ ID NO:28 or SEQ ID NO:32, and/or SEQ ID NO:14 or SEQ ID
NO:24. In certain embodiments, the polypeptide comprises a variable
light chain sequence comprising SEQ ID NO:8 and a variable heavy
chain sequence comprising SEQ ID NO:14. In certain embodiments, the
polypeptide comprises a variable light chain sequence comprising
SEQ ID NO:28 and a variable heavy chain sequence comprising SEQ ID
NO:24. In certain embodiments, the polypeptide comprises a variable
light chain sequence comprising SEQ ID NO:32 and a variable heavy
chain sequence comprising SEQ ID NO:24. In certain embodiments, the
polypeptide is an antibody. In certain embodiments, the polypeptide
specifically binds a non-ligand binding membrane proximal region of
the extracellular domain of a human Notch1 receptor.
It will be recognized in the art that some amino acid sequences of
the invention can be varied without significant effect of the
structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity. Thus,
the invention further includes variations of the polypeptides which
show substantial activity. Such mutants include deletions,
insertions, inversions, repeats, and type substitutions. Guidance
concerning which amino acid changes are likely to be phenotypically
silent can be found in Bowie, J. U., et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 1990, 247:1306-1310.
Thus, the fragments, derivatives, or analogs of the polypeptides of
the invention can be: (i) one in which one or more of the amino
acid residues are substituted with a conserved or non-conserved
amino acid residue (often a conserved amino acid residue) and such
substituted amino acid residue can or cannot be one encoded by the
genetic code; or (ii) one in which one or more of the amino acid
residues includes a substituent group; or (iii) one in which the
mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol); or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for purification
of the mature polypeptide or a proprotein sequence. Such fragments,
derivatives, and analogs are deemed to be within the scope of the
teachings herein.
Of particular interest are substitutions of charged amino acids
with another charged amino acid and with neutral or negatively
charged amino acids. The latter results in proteins with reduced
positive charge. The prevention of aggregation is highly desirable.
Aggregation of proteins not only results in a loss of activity but
can also be problematic when preparing pharmaceutical formulations,
because they can be immunogenic. (Pinckard et al., Clin. Exp.
Immunol. 1967, 2:331-340; Robbins et al., Diabetes 1987,
36:838-845; Cleland et al. Crit. Rev. Therapeutic Drug Carrier
Systems 1993, 10:307-377).
Of course, the number of amino acid substitutions made depends on
many factors, including those described herein. In certain
embodiments, the number of substitutions for any given polypeptide
will not be more than 50, 40, 30, 25, 20, 15, 10 or 3.
The polypeptides of the present invention include the polypeptides
of SEQ ID NO:14 as well as polypeptides which have at least 90%
similarity (at certain times at least 90% sequence identity) to the
polypeptides of SEQ ID NO:14 and at least 95% similarity (at
certain times at least 95% sequence identity) to the polypeptides
of SEQ ID NOs:14, and in still other embodiments, polypeptide which
have at least 96%, 97%, 98%, or 99% similarity (at certain times
96%, 97%, 98%, or 99% sequence identity) to the polypeptides of SEQ
ID NOs:14. The polypeptides of the present invention include the
polypeptides of SEQ ID NO:8 as well as polypeptides which have at
least 90% similarity (at certain times at least 90% sequence
identity) to the polypeptides of SEQ ID NO:8 and at least 95%
similarity (at certain times at least 95% sequence identity) to the
polypeptides of SEQ ID NOs:8, and in still other embodiments,
polypeptide which have at least 96%, 97%, 98%, or 99% similarity
(at certain times 96%, 97%, 98%, or 99% sequence identity) to the
polypeptides of SEQ ID NOs:8. As known in the art "similarity"
between two polypeptides is determined by comparing the amino acid
sequence and its conserved amino acid substitutes of one
polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention
can be employed for producing the corresponding full-length
polypeptide by peptide synthesis; therefore, the fragments can be
employed as intermediates for producing the full-length
polypeptides. Fragments or portions of the polynucleotides of the
present invention can be used to synthesize full-length
polynucleotides of the present invention.
In certain embodiments, a fragment of the proteins of this
invention is a portion or all of a protein which is capable of
binding to a Notch1 receptor protein. This fragment has a high
affinity for a Notch receptor or a ligand of a Notch1 receptor.
Certain fragments of fusion proteins are protein fragments
comprising at least part of the Notch binding domain of the
polypeptide agent or antagonist fused to at least part of a
constant region of an immunoglobulin. The affinity is typically in
the range of about 10.sup.-11 to 10.sup.-12 M, although the
affinity can vary considerably with fragments of different sizes,
ranging from 10.sup.-7 to 10.sup.-13 M. In some embodiments, the
fragment is about 10-110 amino acids in length and comprises the
Notch binding domain of the polypeptide agent or antagonist linked
to at least part of a constant region of an immunoglobulin.
The polypeptides and analogs can be further modified to contain
additional chemical moieties not normally part of the protein. The
derivatized moieties can improve the solubility, the biological
half life and/or absorption of the protein. The moieties can also
reduce or eliminate any undesirable side effects of the protein and
the like. An overview for chemical moieties can be found in
Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co.,
Easton, Pa. (2000).
The isolated polypeptides described herein can be produced by any
suitable method known in the art. Such methods range from direct
protein synthesis methods to constructing a DNA sequence encoding
isolated polypeptide sequences and expressing those sequences in a
suitable transformed host.
In some embodiments of a recombinant method, a DNA sequence is
constructed by isolating or synthesizing a DNA sequence encoding a
wild-type protein of interest. Optionally, the sequence can be
mutagenized by site-specific mutagenesis to provide functional
analogs thereof. See, e.g. Zoeller et al., Proc.-Nat Acad. Sci. USA
1984, 81:5662-5066 and U.S. Pat. No. 4,588,585. Another method of
constructing a DNA sequence encoding a polypeptide of interest
would be by chemical synthesis using an oligonucleotide
synthesizer. Such oligonucleotides can be designed based on the
amino acid sequence of the desired polypeptide and selecting those
codons that are favored in the host cell in which the recombinant
polypeptide of interest will be produced.
Standard methods can be applied to synthesize an isolated
polynucleotide sequence encoding an isolated polypeptide of
interest. For example, a complete amino acid sequence can be used
to construct a back-translated gene. Further, a DNA oligomer
containing a nucleotide sequence coding for the particular isolated
polypeptide can be synthesized. For example, several small
oligonucleotides coding for portions of the desired polypeptide can
be synthesized and then ligated. The individual oligonucleotides
typically contain 5' or 3' overhangs for complementary
assembly.
Once assembled (by synthesis, site-directed mutagenesis, or another
method), the mutant DNA sequences encoding a particular isolated
polypeptide of interest will be inserted into an expression vector
and operatively linked to an expression control sequence
appropriate for expression of the protein in a desired host. Proper
assembly can be confirmed by nucleotide sequencing, restriction
mapping, and expression of a biologically active polypeptide in a
suitable host. As is well known in the art, in order to obtain high
expression levels of a transfected gene in a host, the gene is
operatively linked to transcriptional and translational expression
control sequences that are functional in the chosen expression
host.
The present invention provides isolated antibodies against a
non-ligand binding membrane proximal region of the extracellular
domain of a Notch1 receptor. The antibody, or antibody fragment,
can be any monoclonal or polyclonal antibody that specifically
recognizes a membrane proximal region of the extracellular domain
of Notch1. In some embodiments, the present invention provides
monoclonal antibodies, or fragments thereof, that specifically bind
to a membrane proximal region of the extracellular domain of a
human Notch1 as described herein. In some embodiments, the
monoclonal antibodies, or fragments thereof, are chimeric or
humanized antibodies that specifically bind to a membrane proximal
region of the extracellular domain of a human Notch1 receptor as
described herein. In other embodiments, the monoclonal antibodies,
or fragments thereof, are human antibodies that specifically bind
to a membrane proximal region of the extracellular domain of a
human Notch1 receptor as described herein.
The antibodies against a membrane proximal region of the
extracellular domain of a Notch1 receptor find use in the
experimental, diagnostic and therapeutic methods described herein.
In certain embodiments, the antibodies of the present invention are
used to detect the expression of a Notch1 receptor in biological
samples such as, for example, a patient tissue biopsy, pleural
effusion, or blood sample. Tissue biopsies can be sectioned and
protein detected using, for example, immunofluorescence or
immunohistochemistry. Alternatively, individual cells from a sample
are isolated, and protein expression detected on fixed or live
cells by FACS analysis. Furthermore, the antibodies can be used on
protein arrays to detect expression of a Notch1 receptor, for
example, on tumor cells, in cell lysates, or in other protein
samples. In other embodiments, the antibodies of the present
invention are used to inhibit the growth of tumor cells by
contacting the antibodies with tumor cells either in in vitro cell
based assays or in vivo animal models. In still other embodiments,
the antibodies are used to treat cancer in a human patient by
administering a therapeutically effective amount of an antibody
against a membrane proximal region of the extracellular domain of a
Notch1 receptor.
Polyclonal antibodies can be prepared by any known method.
Polyclonal antibodies are raised by immunizing an animal (e.g. a
rabbit, rat, mouse, goat, donkey, etc.) by multiple subcutaneous or
intraperitoneal injections of the relevant antigen (a purified
peptide fragment, full-length recombinant protein, fusion protein,
etc.) optionally conjugated to keyhole limpet hemocyanin (KLH),
serum albumin, etc. diluted in sterile saline and combined with an
adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form a
stable emulsion. The polyclonal antibody is then recovered from
blood, ascites and the like, of an animal so immunized. Collected
blood is clotted, and the serum decanted, clarified by
centrifugation, and assayed for antibody titer. The polyclonal
antibodies can be purified from serum or ascites according to
standard methods in the art including affinity chromatography,
ion-exchange chromatography, gel electrophoresis, dialysis,
etc.
Monoclonal antibodies can be prepared using hybridoma methods, such
as those described by Kohler and Milstein, 1975, Nature
256:495-497. Using the hybridoma method, a mouse, hamster, or other
appropriate host animal, is immunized as described above to elicit
the production of antibodies by lymphocytes that will specifically
bind to an immunizing antigen. Alternatively, lymphocytes can be
immunized in vitro. Following immunization, the lymphocytes are
isolated and fused with a suitable myeloma cell line using, for
example, polyethylene glycol, to form hybridoma cells that can then
be selected away from unfused lymphocytes and myeloma cells.
Hybridomas that produce monoclonal antibodies directed specifically
against a chosen antigen as determined by immunoprecipitation,
immunoblotting, or by an in vitro binding assay such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)
can then be propagated either in vitro culture using standard
methods (Goding, Monoclonal Antibodies: Principles and Practice,
Academic Press, 1986) or in vivo as ascites tumors in an animal.
The monoclonal antibodies can then be purified from the culture
medium or ascites fluid as described for polyclonal antibodies
above.
In some embodiments of the present invention, the antibody is an
antibody that specifically binds to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor. In some embodiments, the antibody comprises a heavy chain
variable region having at least 90% sequence identity to SEQ ID
NO:14; and/or a light chain variable region having at least 90%
sequence identity to SEQ ID NO:8. In some embodiments, the antibody
comprises a heavy chain variable region having at least 95%
sequence identity to SEQ ID NO:14, and/or a light chain variable
region having at least 95% sequence identity to SEQ ID NO:8. In
some embodiments, the antibody is a monoclonal antibody or antibody
fragment.
In certain embodiments, the invention provides an antibody that
binds a non-ligand binding membrane proximal region of the
extracellular domain of a human Notch1 and comprises a heavy chain
CDR1 comprising RGYWIE (SEQ ID NO:15), a heavy chain CDR2
comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16), and/or a heavy chain
CDR3 comprising FDGNYGYYAMDY (SEQ ID NO:17). In some embodiments,
the antibody further comprises a light chain CDR1 comprising
RSSTGAVTTSNYAN (SEQ ID NO:18), a light chain CDR2 comprising
GTNNRAP (SEQ ID NO:19), and/or a light chain CDR3 comprising
ALWYSNHWVFGGGTKL (SEQ ID NO:20). In some embodiments, the antibody
comprises a heavy chain CDR1 comprising RGYWIE (SEQ ID NO:15), a
heavy chain CDR2 comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16),
and/or a heavy chain CDR3 comprising FDGNYGYYAMDY (SEQ ID NO:17);
and a light chain CDR1 comprising RSSTGAVTTSNYAN (SEQ ID NO:18), a
light chain CDR2 comprising GTNNRAP (SEQ ID NO:19), and/or a light
chain CDR3 comprising ALWYSNHWVFGGGTKL (SEQ ID NO:20). In some
embodiments, the antibody comprises a heavy chain variable region
comprising: (a) a heavy chain CDR1 comprising RGYWIE (SEQ ID
NO:15), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions; (b) a heavy chain CDR2 comprising QILPGTGRTNYNEKFKG
(SEQ ID NO:16), or a variant thereof comprising 1, 2, 3, or 4 amino
acid substitutions; and/or (c) a heavy chain CDR3 comprising
FDGNYGYYAMDY (SEQ ID NO:17), or a variant thereof comprising 1, 2,
3, or 4 amino acid substitutions. In other embodiments, the
antibody comprises a light chain variable region comprising: (a) a
light chain CDR1 comprising RSSTGAVTTSNYAN (SEQ ID NO:18), or a
variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;
(b) a light chain CDR2 comprising GTNNRAP(SEQ ID NO:19), or a
variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;
and/or (c) a light chain CDR3 comprising ALWYSNHWVFGGGTKL (SEQ ID
NO:20), or a variant thereof comprising 1, 2, 3, or 4 amino acid
substitutions. In some embodiments, the amino acid substitutions
are conservative amino acid substitutions.
In some embodiments, the invention provides an antibody, 52M51,
produced by the hybridoma cell line deposited with the ATCC under
the conditions of the Budapest Treaty on Aug. 7, 2008 and assigned
number PTA-9405. In some embodiments, the antibody is a humanized
version of 52M51. In some embodiments, the antibody is a humanized
version of 52M51, "52M51H4L3", as encoded by the DNA deposited with
the ATCC under the conditions of the Budapest Treaty on Oct. 15,
2008 and assigned number PTA-9549. In some embodiments, the
antibody is a humanized version of 52M51, "52M51H4L4". In some
embodiments, the invention provides an antibody that binds to the
same epitope as the epitope to which antibody 52M51 binds. In other
embodiments, the invention provides an antibody that competes with
any of the antibodies as described in the aforementioned
embodiments and/or aspects, as well as other aspects/embodiments
described elsewhere herein, for specific binding to a non-ligand
binding membrane proximal region of the extracellular domain of a
human Notch1 receptor. Pharmaceutical compositions comprising the
antibodies and methods of treating cancer comprising administering
therapeutically effective amounts of the antibodies are also
provided.
In some embodiments, the invention provides an antibody, 52R43, as
encoded by the DNA deposited with the ATCC under the conditions of
the Budapest Treaty on Oct. 15, 2008 and assigned number PTA-9548.
In some embodiments, the invention provides an antibody that binds
to the same epitope as the epitope to which antibody 52R43 binds.
In some embodiments, the invention provides an antibody that
comprises one, two, three, four, five and/or six of the CDRs of
52R43. In other embodiments, the invention provides an antibody
that competes with 52R43. Pharmaceutical compositions comprising
the antibodies and methods of treating cancer comprising
administering therapeutically effective amounts of the antibodies
are also provided.
Alternatively monoclonal antibodies can also be made using
recombinant DNA methods as described in U.S. Pat. No. 4,816,567.
Polynucleotides encoding a monoclonal antibody are isolated from
mature B-cells or hybridoma cells, such as by RT-PCR using
oligonucleotide primers that specifically amplify the genes
encoding the heavy and light chains of the antibody, and their
sequence is determined using conventional procedures. Isolated
polynucleotides encoding the heavy and light chains are then cloned
into suitable expression vectors are then transfected into host
cells such as E. coli cells, simian COS cells, Chinese hamster
ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein. Host cells are screened for monoclonal
antibody production and antibodies with the desired specificity are
selected. Also, recombinant monoclonal antibodies or fragments
thereof of the desired species can be isolated from phage display
libraries as described (McCafferty et al., 1990, Nature,
348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks
et al., 1991, J. Mol. Biol., 222:581-597).
The polynucleotide(s) encoding a monoclonal antibody can further be
modified in a number of different manners using recombinant DNA
technology to generate alternative antibodies. In some embodiments,
the constant domains of the light and heavy chains of, for example,
a mouse monoclonal antibody can be substituted 1) for those regions
of, for example, a human antibody to generate a chimeric antibody
or 2) for a non-immunoglobulin polypeptide to generate a fusion
antibody. In other embodiments, the constant regions are truncated
or removed to generate the desired antibody fragment of a
monoclonal antibody. Furthermore, site-directed or high-density
mutagenesis of the variable region can be used to optimize
specificity, affinity, etc. of a monoclonal antibody.
More generally, modified antibodies useful in the present invention
may be obtained or derived from any antibody. Further, the parent
or precursor antibody, or fragment thereof, used to generate the
disclosed modified antibodies may be murine, human, chimeric,
humanized, non-human primate or primatized. In other embodiments
the modified antibodies of the present invention can comprise
single chain antibody constructs (such as that disclosed in U.S.
Pat. No. 5,892,019, which is incorporated herein by reference)
having altered constant domains as described herein. Consequently,
any of these types of antibodies modified in accordance with the
teachings herein are compatible with this invention.
According to the present invention, techniques can be adapted for
the production of single-chain antibodies specific to a polypeptide
of the invention (see U.S. Pat. No. 4,946,778). In addition,
methods can be adapted for the construction of Fab expression
libraries (Huse, et al., 1989, Science, 246:1275-1281) to allow
rapid and effective identification of monoclonal Fab fragments with
the desired specificity for Notch or derivatives, fragments,
analogs or homologs thereof. Antibody fragments that contain the
idiotypes to a polypeptide of the invention may be produced by
techniques in the art including, but not limited to: (a) an
F(ab').sub.2 fragment produced by pepsin digestion of an antibody
molecule; (b) an Fab fragment generated by reducing the disulfide
bridges of an F(ab').sub.2 fragment, (c) an Fab fragment generated
by the treatment of the antibody molecule with papain and a
reducing agent, and (d) Fv fragments.
Bispecific antibodies are also within the scope of the invention.
Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens.
Methods for making bispecific antibodies are known in the art. For
example, in the present case, one of the binding specificities is
for an antigenic polypeptide of the invention (Notch1 or a fragment
thereof), while the second binding target is any other antigen, and
advantageously is a cell surface protein, or receptor or receptor
subunit. Recombinant production of bispecific antibodies is based
on the co-expression of two immunoglobulin heavy chain/light chain
pairs, where the two heavy chains have different specificities
(Milstein and Cuello, Nature 1983, 305:537-539). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of ten different
antibody molecules, of which only one has the correct bispecific
structure. The purification of the correct molecule is usually
accomplished by affinity chromatography.
Antibody variable domains with the desired binding specificities
can be fused to immunoglobulin constant domain sequences. The
fusion is with an immunoglobulin heavy chain constant domain,
comprising at least part of the hinge, CH2 and CH3 regions. The
first heavy chain constant region (CH1) containing the site
necessary for light chain binding can be present in at least one of
the fusions. DNA encoding the immunoglobulin heavy chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. Further details of generating bispecific antibodies
can be found in Suresh et al., Methods in Enzymology 1986,
121:210.
Bispecific antibodies can be prepared as full-length antibodies or
antibody fragments. Techniques for generating bispecific antibodies
from antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. In addition, Brennan et al., Science 1985, 229:81 describe
a procedure wherein intact antibodies are proteolytically cleaved
to generate F(ab').sub.2 fragments.
Additionally, Fab' fragments can be directly recovered from E. coli
and chemically coupled to form bispecific antibodies (Shalaby et
al., J. Exp. Med. 1992, 175:217-225). These methods can be used in
the production of a fully humanized bispecific antibody
F(ab').sub.2 molecule.
Antibodies with more than two valencies are also contemplated. For
example, trispecific antibodies can be prepared (Tutt et al., 1991,
J. Immunol. 147:60).
This invention also encompasses bispecific antibodies that
specifically recognize the membrane proximal region of a
extracellular domain of a Notch1 receptor. Bispecific antibodies
are antibodies that are capable of specifically recognizing and
binding at least two different epitopes. The different epitopes can
either be within the same molecule (e.g. the same Notch1) or on
different molecules such that both, for example, the antibodies can
specifically recognize and bind a Notch1 receptor, as well as, for
example, 1) an effector molecule on a leukocyte such as a T-cell
receptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16) or 2)
a cytotoxic agent as described in detail below. Bispecific
antibodies can be intact antibodies or antibody fragments.
Techniques for making bispecific antibodies are common in the art
(Millstein et al., 1983, Nature, 305:537-539; Brennan et al., 1985,
Science, 229:81; Suresh et al, 1986, Methods in Enzymol., 121:120;
Traunecker et al., 1991, EMBO J., 10:3655-3659; Shalaby et al.,
1992, J. Exp. Med., 175:217-225; Kostelny et al., 1992, J.
Immunol., 148:1547-1553; Gruber et al., 1994, J. Immunol.,
152:5368; and U.S. Pat. No. 5,731,168).
Exemplary bispecific antibodies can bind to two different epitopes,
at least one of which originates in a polypeptide of the invention.
Alternatively, an anti-antigenic arm of an immunoglobulin molecule
can be combined with an arm which binds to a triggering molecule on
a leukocyte such as a T cell receptor molecule (e.g. CD2, CD3,
CD28, or B7), or Fc receptors for IgG so as to focus cellular
defense mechanisms to the cell expressing the particular antigen.
Bispecific antibodies can also be used to direct cytotoxic agents
to cells which express a particular antigen. These antibodies
possess an antigen-binding arm and an arm which binds a cytotoxic
agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or
TETA.
Heteroconjugate antibodies are also within the scope of the present
invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune cells to unwanted cells (U.S. Pat.
No. 4,676,980). It is contemplated that the antibodies can be
prepared in vitro using known methods in synthetic protein
chemistry, including those involving crosslinking agents. For
example, immunotoxins can be constructed using a disulfide exchange
reaction or by forming a thioether bond. Examples of suitable
reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate.
For the purposes of the present invention, it should be appreciated
that modified antibodies can comprise any type of variable region
that provides for the association of the antibody with a membrane
proximal region of the extracellular domain of a Notch1 receptor.
In this regard, the variable region may comprise or be derived from
any type of mammal that can be induced to mount a humoral response
and generate immunoglobulins against the desired tumor associated
antigen. As such, the variable region of the modified antibodies
can be, for example, of human, murine, non-human primate (e.g.
cynomolgus monkeys, macaques, etc.) or lupine origin. In some
embodiments both the variable and constant regions of the modified
immunoglobulins are human. In other embodiments the variable
regions of compatible antibodies (usually derived from a non-human
source) can be engineered or specifically tailored to improve the
binding properties or reduce the immunogenicity of the molecule. In
this respect, variable regions useful in the present invention can
be humanized or otherwise altered through the inclusion of imported
amino acid sequences.
In some embodiments, of the present invention the monoclonal
antibody against a membrane proximal region of the extracellular
domain of a Notch1 receptor is a humanized antibody. Humanized
antibodies are antibodies that contain minimal sequences from
non-human (e.g., murine) antibodies within the variable regions.
Such antibodies are used therapeutically to reduce antigenicity and
HAMA (human anti-mouse antibody) responses when administered to a
human subject. In practice, humanized antibodies are typically
human antibodies with minimum to no non-human sequences. A human
antibody is an antibody produced by a human or an antibody having
an amino acid sequence corresponding to an antibody produced by a
human.
Humanized antibodies can be produced using various techniques known
in the art. An antibody can be humanized by substituting the CDR of
a human antibody with that of a non-human antibody (e.g. mouse,
rat, rabbit, hamster, etc.) having the desired specificity,
affinity, and/or capability (Jones et al., 1986, Nature,
321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen
et al., 1988, Science, 239:1534-1536). The humanized antibody can
be further modified by the substitution of additional residue
either in the Fv framework region and/or within the replaced
non-human residues to refine and optimize antibody specificity,
affinity, and/or capability.
In some embodiments of the present invention, the antibody is a
humanized antibody which specifically binds to a non-ligand binding
membrane proximal region of the extracellular domain of a human
Notch1 receptor. In some embodiments, the antibody comprises a
heavy chain variable region having at least 90% sequence identity
to SEQ ID NO:24; and/or a light chain variable region having at
least 90% sequence identity to SEQ ID NO:28 or SEQ ID NO:32. In
some embodiments, the antibody comprises a heavy chain variable
region having at least 95% sequence identity to SEQ ID NO:24,
and/or a light chain variable region having at least 95% sequence
identity to SEQ ID NO:28 or SEQ ID NO:32.
In some embodiments, the humanized antibody comprises a heavy chain
variable region of SEQ ID NO:24, and a light chain variable region
of SEQ ID NO:28. In some embodiments, the humanized antibody
comprises a heavy chain variable region of SEQ ID NO:24, and a
light chain variable region of SEQ ID NO:32.
Human antibodies can be directly prepared using various techniques
known in the art. Immortalized human B lymphocytes immunized in
vitro or isolated from an immunized individual that produces an
antibody directed against a target antigen can be generated (See,
for example, Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147
(1):86-95; and U.S. Pat. No. 5,750,373). Also, the human antibody
can be selected from a phage library, where that phage library
expresses human antibodies (Vaughan et al., 1996, Nature
Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, 95:6157-6162;
Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al.,
1991, J. Mol. Biol., 222:581). Humanized antibodies can also be
made in transgenic mice containing human immunoglobulin loci that
are capable, upon immunization, of producing the full repertoire of
human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array into such germ-line mutant mice
will result in the production of human antibodies upon antigen
challenge. (See, for example, Jakobovits et al., 1993, Proc. Natl.
Acad. Sci. USA, 90:2551; Jakobovits et al., 1993, Nature,
362:255-258; Bruggemann et al., 1993, Year in Immuno. 7:33; U.S.
Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
and 5,661,016
Alternatively, phage display technology can be used to produce
human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain
genes are cloned in-frame into either a major or minor coat protein
gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B-cell. Phage
display can be performed in a variety of formats. Several sources
of V-gene segments can be used for phage display. A diverse array
of anti-oxazolone antibodies have been isolated from a small random
combinatorial library of V genes derived from the spleens of
immunized mice. A repertoire of V genes from unimmunized human
donors can be constructed and antibodies to a diverse array of
antigens (including self-antigens) can be isolated.
As discussed above, human antibodies may also be generated by in
vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
It will be appreciated that grafting the entire non-human variable
domains onto human constant regions will produce "classic" chimeric
antibodies. In the context of the present application the term
"chimeric antibodies" will be held to mean any antibody wherein the
immunoreactive region or site is obtained or derived from a first
species and the constant region (which may be intact, partial or
modified in accordance with this invention) is obtained from a
second species. In some embodiments, the antigen binding region or
site will be from a non-human source (e.g. mouse) and the constant
region is human. While the immunogenic specificity of the variable
region is not generally affected by its source, a human constant
region is less likely to elicit an immune response from a human
subject than would the constant region from a non-human source.
The variable domains in both the heavy and light chains are altered
by at least partial replacement of one or more CDRs and, if
necessary, by partial framework region replacement and sequence
changing. Although the CDRs may be derived from an antibody of the
same class or even subclass as the antibody from which the
framework regions are derived, it is envisaged that the CDRs will
be derived from an antibody of different class and preferably from
an antibody from a different species. It must be emphasized that it
may not be necessary to replace all of the CDRs with the complete
CDRs from the donor variable region to transfer the antigen binding
capacity of one variable domain to another. Rather, it may only be
necessary to transfer those residues that are necessary to maintain
the activity of the antigen binding site. Given the explanations
set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it
will be well within the art, either by carrying out routine
experimentation or by trial and error testing to obtain a
functional antibody with reduced immunogenicity.
Alterations to the variable region notwithstanding, it will be
appreciated that the modified antibodies of this invention will
comprise antibodies, or immunoreactive fragments thereof, in which
at least a fraction of one or more of the constant region domains
has been deleted or otherwise altered so as to provide desired
biochemical characteristics such as increased tumor localization or
reduced serum half-life when compared with an antibody of
approximately the same immunogenicity comprising a native or
unaltered constant region. In some embodiments, the constant region
of the modified antibodies will comprise a human constant region.
Modifications to the constant region compatible with this invention
comprise additions, deletions or substitutions of one or more amino
acids in one or more domains. That is, the modified antibodies
disclosed herein may comprise alterations or modifications to one
or more of the three heavy chain constant domains (CH1, CH2 or CH3)
and/or to the light chain constant domain (CL). In some embodiments
of the invention modified constant regions wherein one or more
domains are partially or entirely deleted are contemplated. In
other embodiments the modified antibodies will comprise domain
deleted constructs or variants wherein the entire CH2 domain has
been removed (.DELTA.CH2 constructs). In still other embodiments
the omitted constant region domain will be replaced by a short
amino acid spacer (e.g. 10 residues) that provides some of the
molecular flexibility typically imparted by the absent constant
region.
Besides their configuration, it is known in the art that the
constant region mediates several effector functions. For example,
binding of the C1 component of complement to antibodies activates
the complement system. Activation of complement is important in the
opsonization and lysis of cell pathogens. The activation of
complement also stimulates the inflammatory response and can also
be involved in autoimmune hypersensitivity. Further, antibodies
bind to cells via the Fc region, with a Fc receptor site on the
antibody Fc region binding to a Fc receptor (FcR) on a cell. There
are a number of Fc receptors which are specific for different
classes of antibody, including IgG (gamma receptors), IgE (epsilon
receptors), IgA (alpha receptors) and IgM (mu receptors). Binding
of antibody to Fc receptors on cell surfaces triggers a number of
important and diverse biological responses including engulfment and
destruction of antibody-coated particles, clearance of immune
complexes, lysis of antibody-coated target cells by killer cells
(called antibody-dependent cell-mediated cytotoxicity, or ADCC),
release of inflammatory mediators, placental transfer and control
of immunoglobulin production. Although various Fc receptors and
receptor sites have been studied to a certain extent, there is
still much which is unknown about their location, structure and
functioning.
While not limiting the scope of the present invention, it is
believed that antibodies comprising constant regions modified as
described herein provide for altered effector functions that, in
turn, affect the biological profile of the administered antibody.
For example, the deletion or inactivation (through point mutations
or other means) of a constant region domain may reduce Fc receptor
binding of the circulating modified antibody thereby increasing
tumor localization. In other cases it may be that constant region
modifications, consistent with this invention, moderate complement
binding and thus reduce the serum half life and nonspecific
association of a conjugated cytotoxin. Yet other modifications of
the constant region may be used to eliminate disulfide linkages or
oligosaccharide moieties that allow for enhanced localization due
to increased antigen specificity or antibody flexibility.
Similarly, modifications to the constant region in accordance with
this invention may easily be made using well known biochemical or
molecular engineering techniques.
It will be noted that the modified antibodies may be engineered to
fuse the CH3 domain directly to the hinge region of the respective
modified antibodies. In other constructs it may be desirable to
provide a peptide spacer between the hinge region and the modified
CH2 and/or CH3 domains. For example, compatible constructs could be
expressed wherein the CH2 domain has been deleted and the remaining
CH3 domain (modified or unmodified) is joined to the hinge region
with a 5-20 amino acid spacer. Such a spacer may be added, for
instance, to ensure that the regulatory elements of the constant
domain remain free and accessible or that the hinge region remains
flexible. However, it should be noted that amino acid spacers may,
in some cases, prove to be immunogenic and elicit an unwanted
immune response against the construct. Accordingly, any spacer
added to the construct be relatively non-immunogenic or, even
omitted altogether if the desired biochemical qualities of the
modified antibodies may be maintained.
Besides the deletion of whole constant region domains, it will be
appreciated that the antibodies of the present invention may be
provided by the partial deletion or substitution of a few or even a
single amino acid. For example, the mutation of a single amino acid
in selected areas of the CH2 domain may be enough to substantially
reduce Fc binding and thereby increase tumor localization.
Similarly, it may be desirable to simply delete that part of one or
more constant region domains that control the effector function
(e.g. complement CLQ binding) to be modulated. Such partial
deletions of the constant regions may improve selected
characteristics of the antibody (serum half-life) while leaving
other desirable functions associated with the subject constant
region domain intact. Moreover, as alluded to above, the constant
regions of the disclosed antibodies may be modified through the
mutation or substitution of one or more amino acids that enhances
the profile of the resulting construct. In this respect it may be
possible to disrupt the activity provided by a conserved binding
site (e.g. Fc binding) while substantially maintaining the
configuration and immunogenic profile of the modified antibody. Yet
other embodiments may comprise the addition of one or more amino
acids to the constant region to enhance desirable characteristics
such as effector function or provide for more cytotoxin or
carbohydrate attachment. In such embodiments it can be desirable to
insert or replicate specific sequences derived from selected
constant region domains.
In certain embodiments of the invention, it can be desirable to use
an antibody fragment, rather than an intact antibody, to increase
tumor penetration, for example. Various techniques are known for
the production of antibody fragments. Traditionally, these
fragments are derived via proteolytic digestion of intact
antibodies (for example Morimoto et al., 1993, Journal of
Biochemical and Biophysical Methods 24:107-117 and Brennan et al.,
1985, Science, 229:81). However, these fragments are now typically
produced directly by recombinant host cells as described above.
Thus Fab, Fv, and scFv antibody fragments can all be expressed in,
and secreted from, E. coli or other host cells, thus allowing the
production of large amounts of these fragments. Alternatively, such
antibody fragments can be isolated from the antibody phage
libraries discussed herein. The antibody fragment can also be
linear antibodies as described in U.S. Pat. No. 5,641,870, for
example, and can be monospecific or bispecific. Other techniques
for the production of antibody fragments will be apparent to one of
skill in the art.
It can further be desirable, especially in the case of antibody
fragments, to modify an antibody in order to increase its serum
half-life. This can be achieved, for example, by incorporation of a
salvage receptor binding epitope into the antibody fragment by
mutation of the appropriate region in the antibody fragment or by
incorporating the epitope into a peptide tag that is then fused to
the antibody fragment at either end or in the middle (e.g., by DNA
or peptide synthesis).
The present invention further embraces variants and equivalents
which are substantially homologous to the chimeric, humanized and
human antibodies, or antibody fragments thereof, set forth herein.
These can contain, for example, conservative substitution
mutations, i.e. the substitution of one or more amino acids by
similar amino acids. For example, conservative substitution refers
to the substitution of an amino acid with another within the same
general class such as, for example, one acidic amino acid with
another acidic amino acid, one basic amino acid with another basic
amino acid or one neutral amino acid by another neutral amino acid.
What is intended by a conservative amino acid substitution is well
known in the art.
The invention also pertains to immunoconjugates comprising an
antibody conjugated to a cytotoxic agent. Cytotoxic agents include
chemotherapeutic agents, growth inhibitory agents, toxins (e.g., an
enzymatically active toxin of bacterial, fungal, plant, or animal
origin, or fragments thereof), radioactive isotopes (i.e., a
radioconjugate), etc. Chemotherapeutic agents useful in the
generation of such immunoconjugates include, for example,
methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents.
Enzymatically active toxins and fragments thereof that can be used
include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
Momordica charantia inhibitor, curcin, crotin, Sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. In some embodiments,
the antibodies can be conjugated to radioisotopes, such as
.sup.90Y, .sup.125I, .sup.131I, .sup.123I, .sup.111In, .sup.105Rh,
.sup.153Sm, .sup.67Cu, .sup.67Ga, .sup.166Ho, .sup.177Lu,
.sup.186Re and .sup.188Re using anyone of a number of well known
chelators or direct labeling. In other embodiments, the disclosed
compositions can comprise antibodies coupled to drugs, prodrugs, or
lymphokines such as interferon. Conjugates of the antibody and
cytotoxic agent are made using a variety of bifunctional
protein-coupling agents such as
N-succinimidyl-3-(2-pyridyidithiol)propionate (SPDP), iminothiolane
(IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HCL), active esters (such as disuccinimidyl suberate),
aldehydes (such as glutareldehyde), bis-azido compounds (such as
bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such
as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such
as tolyene 2,6-diisocyanate), and bis-active fluorine compounds
(such as 1,5-difluoro-2,4-dinitrobenzene). Conjugates of an
antibody and one or more small molecule toxins, such as a
calicheamicin, maytansinoids, a trichothene, and CC1065, and the
derivatives of these toxins that have toxin activity, can also be
used. In some embodiments, the modified antibodies can be complexed
with other immunologically active ligands (e.g., antibodies or
fragments thereof) wherein the resulting molecule binds to both the
neoplastic cell and an effector cell such as a T cell.
Regardless of how useful quantities are obtained, the antibodies of
the present invention can be used in any one of a number of
conjugated (i.e. an immunoconjugate) or unconjugated forms.
Alternatively, the antibodies of this invention can be used in a
nonconjugated or "naked" form to harness the subject's natural
defense mechanisms including complement-dependent cytotoxicity
(CDC) and antibody dependent cellular toxicity (ADCC) to eliminate
the malignant cells. The selection of which conjugated or
unconjugated modified antibody to use will depend of the type and
stage of cancer, use of adjunct treatment (e.g., chemotherapy or
external radiation) and patient condition. It will be appreciated
that one could readily make such a selection in view of the
teachings herein.
Competition assays can be used to determine whether two antibodies
bind the same epitope by recognizing identical or sterically
overlapping epitopes. Any method known to one of skill in the art
for determining competitive binding (such as e.g., the immunoassays
described elsewhere herein) may be used.
The antibodies of the present invention can be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as Biacore analysis, FACS analysis, immunofluorescence,
immunocytochemistry, Western blot analysis, radioimmunoassay,
ELISA, "sandwich" immunoassay, immunoprecipitation assay,
precipitin reaction, gel diffusion precipitin reaction,
immunodiffusion assay, agglutination assay, complement-fixation
assay, immunoradiometric assay, fluorescent immunoassay, and
protein A immunoassay. Such assays are routine and well known in
the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York,
which is incorporated by reference herein in its entirety).
In some embodiments, of the present invention the immunospecificity
of an antibody against a membrane proximal region of the
extracellular domain of a human Notch1 receptor is determined using
ELISA. An ELISA assay comprises preparing antigen, coating wells of
a 96 well microtiter plate with antigen, adding the antibody
against a cancer stem cell marker conjugated to a detectable
compound such as an enzymatic substrate (e.g. horseradish
peroxidase or alkaline phosphatase) to the well, incubating for a
period of time and detecting the presence of the antigen.
Alternatively the antibody against a membrane proximal region of
the extracellular domain of a human Notch1 receptor is not
conjugated to a detectable compound, but instead a second
conjugated antibody that recognizes the antibody against a membrane
proximal region of the extracellular domain of a human Notch1
receptor is added to the well. Further, instead of coating the well
with the antigen, the antibody against a membrane proximal region
of the extracellular domain of a human Notch1 receptor can be
coated to the well and a second antibody conjugated to a detectable
compound can be added following the addition of the antigen to the
coated well. It is known to one of skill in the art what parameters
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art (see e.g. Ausubel et al, eds,
1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley
& Sons, Inc., New York at 11.2.1).
The binding affinity of an antibody to a membrane proximal region
of the extracellular domain of Notch1 receptor and the off-rate of
an antibody-antigen interaction can be determined by competitive
binding assays. One example of a competitive binding assay is a
radioimmunoassay comprising the incubation of labeled antigen (e.g.
.sup.3H or .sup.135I), or fragment or variant thereof, with the
antibody of interest in the presence of increasing amounts of
unlabeled antigen followed by the detection of the antibody bound
to the labeled antigen. The affinity of the antibody against a
membrane proximal region of the extracellular domain of a human
Notch1 receptor and the binding off-rates can be determined from
the data by Scatchard plot analysis. In some embodiments, Biacore
kinetic analysis is used to determine the binding on and off rates
of antibodies against a membrane proximal region of the
extracellular domain of a human Notch1 receptor. Biacore kinetic
analysis comprises analyzing the binding and dissociation of
antibodies from chips with immobilized antigen, for example, Notch1
receptors, on their surface.
In certain embodiments, the invention encompasses isolated
polynucleotides that encode a polypeptide comprising an antibody or
fragment thereof, against a non-ligand binding membrane proximal
region of the extracellular domain of a human Notch1 receptor. The
term "polynucleotide encoding a polypeptide" encompasses a
polynucleotide which includes only coding sequences for the
polypeptide as well as a polynucleotide which includes additional
coding and/or non-coding sequences. The polynucleotides of the
invention can be in the form of RNA or in the form of DNA. DNA
includes cDNA, genomic DNA, and synthetic DNA; and can be
double-stranded or single-stranded, and if single stranded can be
the coding strand or non-coding (anti-sense) strand. The
polynucleotides of the invention can be in the form of RNA or in
the form of DNA, which DNA includes cDNA, genomic DNA, and
synthetic DNA. The DNA can be double-stranded or single-stranded,
and if single stranded can be the coding strand or non-coding
(anti-sense) strand.
The present invention further relates to variants of the
hereinabove described polynucleotides which encode for fragments,
analogs, and derivatives. The variant of the polynucleotide can be
a naturally occurring allelic variant of the polynucleotide or a
non-naturally occurring variant of the polynucleotide.
As hereinabove indicated, the polynucleotide can have a coding
sequence which is a naturally occurring allelic variant of the
coding sequence of the disclosed polypeptides. As known in the art,
an allelic variant is an alternate form of a polynucleotide
sequence which has a substitution, deletion or addition of one or
more nucleotides, which does not substantially alter the function
of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the
coding sequence for the mature polypeptide can be fused in the same
reading frame to a polynucleotide which aids in expression and
secretion of a polypeptide from a host cell, for example, a leader
sequence which functions as a secretory sequence for controlling
transport of a polypeptide from the cell. The polypeptide having a
leader sequence is a preprotein and can have the leader sequence
cleaved by the host cell to form the mature form of the
polypeptide. The polynucleotides can also encode for a proprotein
which is the mature protein plus additional 5' amino acid residues.
A mature protein having a prosequence is a proprotein and is an
inactive form of the protein. Once the prosequence is cleaved an
active mature protein remains. Thus, for example, the
polynucleotide of the present invention can encode for a mature
protein, or for a protein having a prosequence or for a protein
having both a prosequence and presequence (leader sequence).
The polynucleotides of the present invention can also have the
coding sequence fused in frame to a marker sequence which allows
for purification of the polypeptide of the present invention. The
marker sequence can be a hexa-histidine tag supplied by a pQE-9
vector to provide for purification of the mature polypeptide fused
to the marker in the case of a bacterial host, or, for example, the
marker sequence can be a hemagglutinin (HA) tag when a mammalian
host, e.g. COS-7 cells, is used. The HA tag corresponds to an
epitope derived from the influenza hemagglutinin protein (Wilson et
al., 1984, Cell 37:767).
Further embodiments of the invention include isolated nucleic acid
molecules comprising a polynucleotide having a nucleotide sequence
at least 90% identical, 95% identical, and in some embodiments, at
least 96%, 97%, 98% or 99% identical to the disclosed sequences. In
some embodiments, the polynucleotides have a nucleotide sequence at
least 90% identical to SEQ ID NOs: 3, 5, 7, 9, 11, 13, 21, 25 or 29
(with or without signal sequence). In some embodiments, the
polynucleotides have a nucleotide sequence at least 90% identical
to SEQ ID NOs:7 or 13. In some embodiments, the invention provides
a polynucleotide that hybridizes to a polynucleotide encoding the
polypeptides of SEQ ID NOs:4, 6, 8, 10, 12, 14, 22, 23, 24, 26, 27,
28, 30, 31, or 32. In some embodiments, the polynucleotides
hybridize to the polynucleotides of SEQ ID NOs:3, 5, 7, 9, 11, 13,
21, 25 or 29. In some embodiments, the polynucleotides hybridize
under stringent hybridization conditions.
As used herein, the phrases "hybridizes" or "selectively
hybridizes" or "specifically hybridizes" refer to the binding or
duplexing of a molecule only to a particular nucleotide sequence
under stringent hybridization conditions when that sequence is
present in a complex mixture (e.g., a library of DNAs or RNAs).
See, e.g., Andersen (1998) Nucleic Acid Hybridization
Springer-Verlag; Ross (ed. 1997) Nucleic Acid Hybridization
Wiley.
As used herein, the phrase "stringent hybridization conditions"
refers to conditions under which a probe will hybridize to its
target subsequence, typically in a complex mixture of nucleic acid,
but to no other sequences. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Probes, "Overview of principles
of hybridization and the strategy of nucleic acid assays" (1993).
Generally, stringent conditions are selected to be about
5-10.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength. The Tm is the
temperature (under defined ionic strength, pH, and nucleic
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions will be those in
which the salt concentration is less than about 1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g., greater than 50
nucleotides). Stringent conditions can also be achieved with the
addition of destabilizing agents such as formamide. For high
stringency hybridization, a positive signal is at least two times
background, or 10 times background hybridization. Exemplary high
stringency or stringent hybridization conditions include: 50%
formamide, 5.times.SSC, and 1% SDS incubated at 42.degree. C. or
5.times.SSC and 1% SDS incubated at 65.degree. C., with a wash in
0.2.times.SSC and 0.1% SDS at 65.degree. C. For PCR, a temperature
of about 36.degree. C. is typical for low stringency amplification,
although annealing temperatures can vary from about 32.degree. C.
to about 48.degree. C. depending on primer length. For high
stringency PCR amplification, a temperature of about 62.degree. C.
is typical, although high stringency annealing temperatures can
range from about 50.degree. C. to about 65.degree. C., depending on
the primer length and specificity. Typical cycle conditions for
both high and low stringency amplifications include a denaturation
phase of 90.degree. C. to 95.degree. C. for 30-120 sec, an
annealing phase lasting 30-120 sec, and an extension phase of about
72.degree. C. for 1-2 min.
By a polynucleotide having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence is
intended that the nucleotide sequence of the polynucleotide is
identical to the reference sequence except that the polynucleotide
sequence can include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence can be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence can be inserted into
the reference sequence. These mutations of the reference sequence
can occur at the amino- or carboxy-terminal positions of the
reference nucleotide sequence or anywhere between those terminal
positions, interspersed either individually among nucleotides in
the reference sequence or in one or more contiguous groups within
the reference sequence.
As a practical matter, whether any particular nucleic acid molecule
is at least 95%, 96%, 97%, 98% or 99% identical to a reference
sequence can be determined conventionally using known computer
programs such as the Bestfit program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit
uses the local homology algorithm of Smith and Waterman, Advances
in Applied Mathematics 2: 482 489 (1981), to find the best segment
of homology between two sequences. When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference nucleotide sequence and that gaps in
homology of up to 5% of the total number of nucleotides in the
reference sequence are allowed.
The polynucleotide variants can contain alterations in the coding
regions, non-coding regions, or both. In some embodiments the
polynucleotide variants contain alterations which produce silent
substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. In some
embodiments, nucleotide variants are produced by silent
substitutions due to the degeneracy of the genetic code.
Polynucleotide variants can be produced for a variety of reasons,
e.g., to optimize codon expression for a particular host (change
codons in the human mRNA to those preferred by a bacterial host
such as E. coli).
The polypeptides of the present invention can be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides
comprising an antibody, or fragment thereof, against a non-ligand
binding membrane proximal region of the extracellular domain of a
human Notch1 receptor. It will be recognized in the art that some
amino acid sequences of the invention can be varied without
significant effect of the structure or function of the protein.
Thus, the invention further includes variations of the polypeptides
which show substantial activity or which include regions of an
antibody, or fragment thereof, against a membrane proximal region
of the extracellular domain of a human Notch1 receptor. Such
mutants include deletions, insertions, inversions, repeats, and
type substitutions.
The polypeptides and polynucleotides of the present invention are
provided in an isolated form, and at times are purified to
homogeneity.
The isolated polypeptides described herein can be produced by any
suitable method known in the art. Such methods range from direct
protein synthesis methods to constructing a DNA sequence encoding
isolated polypeptide sequences and expressing those sequences in a
suitable transformed host. For example, cDNA can be obtained by
screening a human cDNA library with a labeled DNA fragment encoding
a polypeptide (for example, nucleotide SEQ ID NO:1) and identifying
positive clones by autoradiography. Further rounds of plaque
purification and hybridization are performed using conventional
methods.
In some embodiments of a recombinant method, a DNA sequence is
constructed by isolating or synthesizing a DNA sequence encoding a
wild-type protein of interest. Optionally, the sequence can be
mutagenized by site-specific mutagenesis to provide functional
analogs thereof. (See, e.g. Zoeller et al., 1984, Proc.-Nat Acad.
Sci. USA, 81:5662-5066 and U.S. Pat. No. 4,588,585.) Another method
of constructing a DNA sequence encoding a polypeptide of interest
would be by chemical synthesis using an oligonucleotide
synthesizer. Such oligonucleotides can be designed based on the
amino acid sequence of the desired polypeptide and selecting those
codons that are favored in the host cell in which the recombinant
polypeptide of interest will be produced.
Standard methods can be applied to synthesize an isolated
polynucleotide sequence encoding an isolated polypeptide of
interest. For example, a complete amino acid sequence can be used
to construct a back-translated gene. Further, a DNA oligomer
containing a nucleotide sequence coding for the particular isolated
polypeptide can be synthesized. For example, several small
oligonucleotides coding for portions of the desired polypeptide can
be synthesized and then ligated. The individual oligonucleotides
typically contain 5' or 3' overhangs for complementary
assembly.
Once assembled (by synthesis, site-directed mutagenesis or another
method), the mutant DNA sequences encoding a particular isolated
polypeptide of interest will be inserted into an expression vector
and operatively linked to an expression control sequence
appropriate for expression of the protein in a desired host. Proper
assembly can be confirmed by nucleotide sequencing, restriction
mapping, and expression of a biologically active polypeptide in a
suitable host. As is well known in the art, in order to obtain high
expression levels of a transfected gene in a host, the gene is
operatively linked to transcriptional and translational expression
control sequences that are functional in the chosen expression
host.
Recombinant expression vectors are used to amplify and express DNA
encoding cancer stem cell marker polypeptide fusions. Recombinant
expression vectors are replicable DNA constructs which have
synthetic or cDNA-derived DNA fragments encoding a cancer stem cell
marker polypeptide fusion or a bioequivalent analog operatively
linked to suitable transcriptional or translational regulatory
elements derived from mammalian, microbial, viral or insect genes.
A transcriptional unit generally comprises an assembly of (1) a
genetic element or elements having a regulatory role in gene
expression, for example, transcriptional promoters or enhancers,
(2) a structural or coding sequence which is transcribed into mRNA
and translated into protein, and (3) appropriate transcription and
translation initiation and termination sequences, as described in
detail below. Such regulatory elements can include an operator
sequence to control transcription. The ability to replicate in a
host, usually conferred by an origin of replication, and a
selection gene to facilitate recognition of transformants can
additionally be incorporated. DNA regions are operatively linked
when they are functionally related to each other. For example, DNA
for a signal peptide (secretory leader) is operatively linked to
DNA for a polypeptide if it is expressed as a precursor which
participates in the secretion of the polypeptide; a promoter is
operatively linked to a coding sequence if it controls the
transcription of the sequence; or a ribosome binding site is
operatively linked to a coding sequence if it is positioned so as
to permit translation. Generally, operatively linked means
contiguous and, in the case of secretory leaders, means contiguous
and in reading frame. Structural elements intended for use in yeast
expression systems include a leader sequence enabling extracellular
secretion of translated protein by a host cell. Alternatively,
where recombinant protein is expressed without a leader or
transport sequence, it can include an N-terminal methionine
residue. This residue can optionally be subsequently cleaved from
the expressed recombinant protein to provide a final product.
The choice of expression control sequence and expression vector
will depend upon the choice of host. A wide variety of expression
host/vector combinations can be employed. Useful expression vectors
for eukaryotic hosts, include, for example, vectors comprising
expression control sequences from SV40, bovine papilloma virus,
adenovirus and cytomegalovirus. Useful expression vectors for
bacterial hosts include known bacterial plasmids, such as plasmids
from Esherichia coli, including pCR1, pBR322, pMB9 and their
derivatives, and wider host range plasmids, such as M13 and
filamentous single-stranded DNA phages.
Suitable host cells for expression of a cancer stem cell marker
protein include prokaryotes, yeast, insect or higher eukaryotic
cells. Prokaryotes include gram negative or gram positive
organisms, for example E. coli or bacilli. Higher eukaryotic cells
include established cell lines of mammalian origin as described
below. Cell-free translation systems could also be employed.
Appropriate cloning and expression vectors for use with bacterial,
fungal, yeast, and mammalian cellular hosts are described by
Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier,
N.Y., 1985), the relevant disclosure of which is hereby
incorporated by reference.
Various mammalian or insect cell culture systems are also
advantageously employed to express recombinant protein. Expression
of recombinant proteins in mammalian cells can be performed because
such proteins are generally correctly folded, appropriately
modified and completely functional. Examples of suitable mammalian
host cell lines include the COS-7 lines of monkey kidney cells,
described by Gluzman (1981, Cell, 23:175), and other cell lines
capable of expressing an appropriate vector including, for example,
L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell
lines. Mammalian expression vectors can comprise nontranscribed
elements such as an origin of replication, a suitable promoter and
enhancer linked to the gene to be expressed, and other 5' or 3'
flanking nontranscribed sequences, and 5' or 3' nontranslated
sequences, such as necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites, and
transcriptional termination sequences. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, 1988, Bio/Technology, 6:47.
The proteins produced by a transformed host can be purified
according to any suitable method. Such standard methods include
chromatography (e.g., ion exchange, affinity and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for protein purification. Affinity tags
such as hexahistidine, maltose binding domain, influenza coat
sequence and glutathione-S-transferase can be attached to the
protein to allow easy purification by passage over an appropriate
affinity column. Isolated proteins can also be physically
characterized using such techniques as proteolysis, nuclear
magnetic resonance and x-ray crystallography.
For example, supernatants from systems which secrete recombinant
protein into culture media can be first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. Following the
concentration step, the concentrate can be applied to a suitable
purification matrix. Alternatively, an anion exchange resin can be
employed, for example, a matrix or substrate having pendant
diethylaminoethyl (DEAE) groups. The matrices can be acrylamide,
agarose, dextran, cellulose or other types commonly employed in
protein purification. Alternatively, a cation exchange step can be
employed. Suitable cation exchangers include various insoluble
matrices comprising sulfopropyl or carboxymethyl groups. Finally,
one or more reversed-phase high performance liquid chromatography
(RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica
gel having pendant methyl or other aliphatic groups, can be
employed to further purify a recombinant protein or cancer stem
cell protein-Fc composition. Some or all of the foregoing
purification steps, in various combinations, can also be employed
to provide a homogeneous recombinant protein.
Recombinant protein produced in bacterial culture is usually
isolated by initial extraction from cell pellets, followed by one
or more concentration, salting-out, aqueous ion exchange or size
exclusion chromatography steps. High performance liquid
chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of a recombinant protein can
be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing
agents.
The present invention also provides methods for inhibiting the
growth of tumorigenic cells expressing a cancer stem cell marker
using the antagonists of a cancer stem cell marker described
herein. In some embodiments, the method of inhibiting the growth of
tumorigenic cells expressing a cancer stem cell marker, for example
Notch1 receptor, comprises contacting the cell with an antagonist
against a cancer stem cell marker in vitro. For example, an
immortalized cell line or a cancer cell line that expresses a
cancer stem cell marker is cultured in medium to which is added an
antagonist of the expressed cancer stem cell marker to inhibit cell
growth. In some embodiments, tumor cells comprising tumor stem
cells are isolated from a patient sample such as, for example, a
tissue biopsy, pleural effusion, or blood sample and cultured in
medium to which is added an antagonist of a cancer stem cell marker
to inhibit cell growth. In some embodiments, the antagonist is an
antibody that specifically recognizes an epitope of a cancer stem
cell marker protein. For example, antibodies against a cancer stem
cell marker protein can be added to the culture medium of isolated
cancer stem cells to inhibit cell growth.
In some embodiments, the method of inhibiting the growth of
tumorigenic cells expressing a cancer stem cell marker comprises
contacting the cell with an antagonist against a cancer stem cell
marker in vivo. In some embodiments, the method of inhibiting
growth of tumorigenic cells expressing Notch1 comprises contacting
the cells with an antibody that specifically binds to a non-ligand
binding membrane proximal region of a human Notch1 receptor. In
some embodiments, the antibody inhibits growth of tumorigenic cells
by inhibiting the activity of Notch1. In some embodiments, the
antibody inhibits growth of tumorigenic cells by inhibiting
ligand-induced Notch1 signaling. In some embodiments, the antibody
inhibits growth of tumorigenic cells by inhibiting the cleavage of
Notch1. In some embodiments, the antibody inhibits growth of
tumorigenic cells by reducing the frequency or the number of cancer
stem cells in the tumor.
In certain embodiments, contacting a tumorigenic cell with an
antagonist to a cancer stem cell marker is undertaken in an animal
model. For example, xenografts expressing a cancer stem cell marker
are grown in immunocompromised mice (e.g. NOD/SCID mice) that are
administered an antagonist to a cancer stem cell marker to inhibit
tumor growth. In some embodiments, cancer stem cells that express a
cancer stem cell marker are isolated from a patient sample such as,
for example, a tissue biopsy, pleural effusion, or blood sample and
injected into immunocompromised mice that are then administered an
antagonist against the cancer stem cell marker to inhibit tumor
cell growth. In some embodiments, the antagonist of a cancer stem
cell marker is administered at the same time or shortly after
introduction of tumorigenic cells into the animal to prevent tumor
growth. In other embodiments, the antibody against the cancer stem
cell marker is administered as a therapeutic agent after the
tumorigenic cells have grown to a specified size.
The present invention further provides pharmaceutical compositions
comprising antibodies, polypeptides or other agents that target a
cancer stem cell marker. These pharmaceutical compositions find use
in inhibiting tumor growth, tumor cell growth and treating cancer
in human patients.
Formulations are prepared for storage and use by combining a
purified antagonist (e.g., antibody) of the present invention with
a pharmaceutically acceptable vehicle (e.g., carrier, excipient,
etc.) (Remington, The Science and Practice of Pharmacy 20th Edition
Mack Publishing, 2000). Suitable pharmaceutically acceptable
vehicles include, but are not limited to, nontoxic buffers such as
phosphate, citrate, and other organic acids; salts such as sodium
chloride; antioxidants including ascorbic acid and methionine;
preservatives such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride; benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol; low molecular weight polypeptides (less
than about 10 amino acid residues); proteins such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; carbohydrates such as
monosacchandes, disaccharides, glucose, mannose, or dextrins;
chelating agents such as EDTA; sugars such as sucrose, mannitol,
trehalose or sorbitol; salt-forming counter-ions such as sodium;
metal complexes such as Zn-protein complexes; and/or non-ionic
surfactants such as TWEEN or polyethylene glycol (PEG).
The pharmaceutical composition of the present invention can be
administered in any number of ways for either local or systemic
treatment. Administration can be topical (such as to mucous
membranes including vaginal and rectal delivery) such as
transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders; pulmonary such as by
inhalation or insufflation of powders or aerosols (including by
nebulizer), intratracheal, intranasal, epidermal and transdermal;
oral; parenteral including intravenous, intraarterial,
intratumoral, subcutaneous, intraperitoneal or intramuscular
injection or infusion; or intracranial such asintrathecal or
intraventricular.
The therapeutic formulation can be in unit dosage form. Such
formulations include tablets, pills, capsules, powders, granules,
solutions or suspensions in water or non-aqueous media, or
suppositories for oral, parenteral, or rectal administration or for
administration by inhalation. In solid compositions such as tablets
the principal active ingredient is mixed with a pharmaceutical
carrier. Conventional tableting ingredients include corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other diluents (e.g., water) to
form a solid preformulation composition containing a homogeneous
mixture of a compound of the present invention, or a non-toxic
pharmaceutically acceptable salt thereof. The solid preformulation
composition is then subdivided into unit dosage forms of the type
described herein. The tablets, pills, etc of the novel composition
can be coated or otherwise compounded to provide a dosage form
affording the advantage of prolonged action. For example, the
tablet or pill can comprise an inner composition covered by an
outer component. Furthermore, the two components can be separated
by an enteric layer that serves to resist disintegration and
permits the inner component to pass intact through the stomach or
to be delayed in release. A variety of materials can be used for
such enteric layers or coatings, such materials including a number
of polymeric acids and mixtures of polymeric acids with such
materials as shellac, cetyl alcohol and cellulose acetate.
Pharmaceutical formulations include antibodies of the present
invention complexed with liposomes (Epstein, et al., 1985, Proc.
Natl. Acad. Sci. USA, 82:3688; Hwang, et al., 1980, Proc. Natl.
Acad. Sci. USA, 77:4030; and U.S. Pat. Nos. 4,485,045 and
4,544,545). Liposomes with enhanced circulation time are disclosed
in U.S. Pat. No. 5,013,556. Some liposomes can be generated by the
reverse phase evaporation with a lipid composition comprising
phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
The antibodies can also be entrapped in microcapsules. Such
microcapsules are prepared, for example, by coacervation techniques
or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions as described in Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing (2000).
In addition sustained-release preparations can be prepared.
Suitable examples of sustained-release preparations include
semi-permeable matrices of solid hydrophobic polymers containing
the antibody, which matrices are in the form of shaped articles
(e.g. films, or microcapsules). Examples of sustained-release
matrices include polyesters, hydrogels such as
poly(2-hydroxyethyl-methacrylate) or poly(v nylalcohol),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D(-)-3-hydroxybutyric acid. In some
embodiments the antibodies can be used to treat various conditions
characterized by expression and/or increased responsiveness of
cells to a cancer stem cell marker. Particularly it is envisioned
that the antibodies against a cancer stem cell marker, for example
Notch1, will be used to treat proliferative disorders including but
not limited to benign and malignant tumors of the kidney, liver,
bladder, breast, stomach, ovary, colon, rectum, prostate, lung,
vulva, thyroid, head and neck, brain (glioblastoma, astrocytoma,
medulloblastoma, etc), blood and lymph (leukemias and
lymphomas).
In some embodiments, the treatment involves the combined
administration of an antibody or other agent of the present
invention and a chemotherapeutic agent or cocktail of multiple
different chemotherapeutic agents. Treatment with an antibody can
occur prior to, concurrently with, or subsequent to administration
of chemotherapies. Chemotherapies contemplated by the invention
include chemical substances or drugs which are known in the art and
are commercially available, such as doxorubicin, 5-fluorouracil,
cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa,
busulfan, cytoxin, taxol, methotrexate, cisplatin, melphalan,
vinblastine and carboplatin. Combined administration can include
co-administration, either in a single pharmaceutical formulation or
using separate formulations, or consecutive administration in
either order but generally within a time period such that all
active agents can exert their biological activities simultaneously.
Preparation and dosing schedules for such chemotherapeutic agents
can be used according to manufacturers' instructions or as
determined empirically. Preparation and dosing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992).
Chemotherapeutic agents useful in the instant invention also
include, but are not limited to, alkylating agents such as thiotepa
and cyclophosphamide (Cytoxan); alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide and
trimethylolomelamime nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,
chromomycins, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine, 5-FU; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic
acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan;
vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide;
thiotepa; taxoids, e.g. paclitaxel (TAXOL, Bristol-Myers Squibb
Oncology, Princeton, N.J.) and doxetaxel (Taxotere, Rhone-Poulenc
Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;
navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda;
ibandronate; CPT11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoic acid; esperamicins;
capecitabine; and pharmaceutically acceptable salts, acids or
derivatives of any of the above. Chemotherapeutic agents also
include anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and toremifene (Fareston); and antiandrogens
such as flutamide, nilutamide, bicalutamide, leuprolide, and
goserelin; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
In certain embodiments, the chemotherapeutic agent is a
topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy
agents that interfere with the action of a topoisomerase enzyme
(e.g., topoisomerase I or II). Topoisomerase inhibitors include,
but are not limited to, doxorubicin HCL, daunorubicin citrate,
mitoxantrone HCL, actinomycin D, etoposide, topotecan HCL,
teniposide (VM-26), and irinotecan.
In certain embodiments, the chemotherapeutic agent is an
anti-metabolite. An anti-metabolite is a chemical with a structure
that is similar to a metabolite required for normal biochemical
reactions, yet different enough to interfere with one or more
normal functions of cells, such as cell division. Anti-metabolites
include, but are not limited to, gemcitabine, fluorouracil,
capecitabine, methotrexate sodium, ralitrexed, Pemetrexed, tegafur,
cytosine arabinoside, Thioguanine (GlaxoSmithKline), 5-azacytidine,
6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin,
fludarabine phosphate, and cladribine, as well as pharmaceutically
acceptable salts, acids, or derivatives of any of these.
In other embodiments, the treatment involves the combined
administration of an antibody or other agent of the present
invention and radiation therapy. Treatment with an antibody can
occur prior to, concurrently with, or subsequent to administration
of radiation therapy. Any dosing schedules for such radiation
therapy can be used as determined by the skilled practitioner.
In other embodiments, the treatment can involve the combined
administration of antibodies of the present invention with other
antibodies against additional tumor associated antigens including,
but not limited to, antibodies that bind to the EGF receptor (EGFR)
(Erbitux.RTM.), the erbB2 receptor (HER2) (Herceptin.RTM.), and
vascular endothelial growth factor (VEGF) (Avastin.RTM.).
Furthermore, treatment can include administration of one or more
cytokines; can be accompanied by surgical removal of cancer cells;
and/or any other therapy deemed necessary by a treating
physician.
For the treatment of the disease, the appropriate dosage of an
antibody or other agent of the present invention depends on the
type of disease to be treated, the severity and course of the
disease, the responsiveness of the disease, whether the antibody is
administered for therapeutic or preventative purposes, previous
therapy, patient's clinical history, and so on all at the
discretion of the treating physician. The antibody or agent can be
administered one time or over a series of treatments lasting from
several days to several months, or until a cure is effected or a
diminution of the disease state is achieved (e.g. reduction in
tumor size). Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient and
will vary depending on the relative potency of an individual
antagonist. The administering physician can easily determine
optimum dosages, dosing methodologies and repetition rates. In
general, dosage is from 0.01 .mu.g to 100 mg per kg of body weight,
and can be given once or more daily, weekly, monthly or yearly. The
treating physician can estimate repetition rates for dosing based
on measured residence times and concentrations of the antibody or
agent in bodily fluids or tissues.
The present invention provides kits comprising the antibodies
described herein and that can be used to perform the methods
described herein. In some embodiments, a kit comprises at least one
purified antibody against a cancer stem cell marker, in one or more
containers. In some embodiments, a kit comprises at least one
purified antibody against a non-ligand binding membrane proximal
region of the extracellular domain of a human Notch1 receptor, in
one or more containers. In some embodiments, a kit comprises the
antibody 52M51 or a humanized variant of 52M51. In some
embodiments, a kit comprises the antibody 52R43. In some
embodiments, the kits contain all of the components necessary
and/or sufficient to perform a detection assay, including all
controls, directions for performing assays, and any necessary
software for analysis and presentation of results. One skilled in
the art will readily recognize that the disclosed antibodies of the
present invention can be readily incorporated into one of the
established kit formats which are well known in the art.
In certain embodiments, the present invention provides a method of
identifying a molecule that binds to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor and inhibits tumor growth, the method comprising: i)
incubating the molecule with the non-ligand binding membrane
proximal region of the extracellular domain of the human Notch1
receptor; ii) determining if the molecule binds to the membrane
proximal region of the extracellular domain of the human Notch
receptor; and iii) determining if the molecule inhibits tumor
growth. Molecules that specifically bind a membrane proximal region
of the extracellular domain of a human Notch1 receptor include, but
are not limited to, polypeptides and antibodies.
Screening can be performed using any suitable method known in the
art. In certain embodiments, screening is performed in vitro. In
some embodiments, cells expressing a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor are incubated with a labeled molecule and specific binding
of the labeled molecule to a membrane proximal region of the
extracellular domain of a human Notch1 receptor is determined by
FACS analysis. In some embodiments, a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor is expressed by phage display, and molecules that
specifically binding to a membrane proximal region of the
extracellular domain of a human Notch1 receptor are identified.
Other suitable methods for identifying molecules that specifically
bind to a non-ligand binding membrane proximal region of a human
Notch1 receptor include, but are not limited to, ELISA; Western (or
immuno) blotting; and yeast-two-hybrid.
Molecules that specifically bind to a non-ligand binding membrane
proximal region of the extracellular domain of a human Notch1
receptor are then tested for inhibition of tumor cell growth.
Testing can be performed using any suitable method known in the
art. In certain embodiments, molecules that specifically bind to
membrane proximal region of the extracellular domain of a human
Notch1 receptor are tested for the ability to inhibit tumor growth
in vitro. In some embodiments, molecules that specifically bind a
membrane proximal region of the extracellular domain of a human
Notch1 receptor are incubated with tumor cells in culture and
proliferation of tumor cells in the presence of the molecule that
specifically binds a membrane proximal region of the extracellular
domain of a human Notch1 receptor is determined and compared to
tumor cells incubated with a non-binding control molecule. In
certain embodiments, molecules that specifically bind to non-ligand
binding membrane proximal region of the extracellular domain of a
human Notch1 receptor are tested for the ability to inhibit tumor
growth in vivo. In certain embodiments, molecules that specifically
bind a membrane proximal region of the extracellular domain of a
human Notch1 receptor are injected into an animal xenograft model
and the growth of tumors in animals treated with molecules that
specifically bind to the membrane proximal region of the
extracellular domain of a human Notch1 receptor is determined and
compared to animals treated with a non-binding control
molecule.
EXAMPLES
Example 1
Antibodies were generated against a non-ligand binding region of
Notch1, specifically the non-ligand binding membrane proximal
region of the extracellular domain. In certain embodiments,
recombinant polypeptide fragments of the human Notch1 extracellular
domain were generated as antigens for antibody production. Standard
recombinant DNA technology was used to isolate polynucleotides
encoding the membrane proximal region of the extracellular domain
of human Notch1 amino acids 1427-1732 (SEQ ID NO:1). These
polynucleotides were separately ligated in-frame N-terminal to a
human Fc and histidine-tag and cloned into a transfer plasmid
vector for baculovirus-mediated expression in insect cells.
Standard transfection, infection, and cell culture protocols were
used to produce recombinant insect cells expressing the
corresponding Notch1 polypeptide corresponding to a membrane
proximal region comprising amino acids 1427-1732 (SEQ ID NO:2)
(O'Reilly et al., 1994, Baculovirus Expression Vectors: A
Laboratory Manual, Oxford: Oxford University Press).
Notch1 membrane proximal region (Notch1 amino acids 1472-1732)
polypeptide was purified from insect cell lysates using protein A
and Ni++-chelate affinity chromatography as known to one skilled in
the art. Purified Notch1 membrane proximal region polypeptide was
dialyzed against PBS (pH=7), concentrated to approximately 1 mg/ml,
and sterile filtered in preparation for immunization.
Mice (n=3) were immunized with purified Notch1 antigen protein
(Antibody Solutions; Mountain View, Calif.) using standard
techniques. Blood from individual mice was screened approximately
70 days after initial immunization for antigen recognition using
ELISA and FACS analysis (as described herein). The two animals with
the highest antibody titers were selected for final antigen boost
after which spleen cells were isolated for hybridoma production.
Hybridoma cells were plated at 1 cell per well in 96 well plates,
and the supernatant from each well screened by ELISA and FACS
analysis against Notch1 membrane proximal region polypeptide.
Several hybridomas with high antibody titer were selected and
scaled up in static flask culture. Antibodies were purified from
the hybridoma supernatant using protein A or protein G agarose
chromatography. Purified monoclonal antibodies were tested again by
FACS as described herein. Several antibodies that recognized the
membrane proximal region of the extracellular domain of human
Notch1 were isolated. A hydridoma cell line expressing antibody
52M51 was deposited with ATCC under the conditions of the Budapest
Treaty on Aug. 7, 2008 and assigned ATTC Patent Deposit Designation
PTA-9405. The nucleotide and predicted protein sequences of both
the heavy chain (SEQ ID NO:9 and 10) and light chain (SEQ ID NO:3
and 4) of antibody 52M51 were determined.
Human Antibodies
In alternative embodiments, human antibodies that specifically
recognize the non-ligand binding membrane proximal region of the
extracellular domain of a Notch1 receptor are isolated using phage
display technology. In certain embodiments, a synthetic antibody
library containing human antibody variable domains is screened for
specific and high affinity recognition of a Notch receptor antigen
described herein. In certain embodiments, a human Fab phage display
library is screened using a series of recombinant proteins
comprising the non-ligand binding membrane proximal region of the
extracellular domain of a Notch 1 receptor. Briefly,
2.times.10.sup.13 Fab displaying phage particles are incubated with
recombinant protein (passively immobilized) in round one, the
non-specific phage are washed off, and then specific phage are
eluted with either low pH (cells) or DTT (recombinant protein). The
eluted output is used to infect TG1 F+ bacteria, rescued with
helper phage, and then Fab display induced with IPTG (0.25 mM).
This process is repeated for two additional rounds and then round
three is screened in ELISA against passively immobilized antigen (5
.mu.g/ml).
CDR cassettes in the library are specifically exchanged via unique
flanking restriction sites for antibody optimization. Optimized
human variable regions are then cloned into an Ig expression vector
containing human IgG1 heavy-chain and kappa light-chain for
expression of human antibodies in CHO cells.
Epitope Mapping
To identify antibodies that recognize specific a non-ligand binding
membrane proximal region of the Notch1 receptor extracellular
domains, epitope mapping is performed. In certain embodiments,
mammalian expression plasmid vectors comprising a CMV promoter
upstream of polynucleotides that encode fragments of the
extracellular Notch1 domain as Fc fusion proteins are generated
using standard recombinant DNA technology. In certain embodiments,
epitope mapping of the 52M series of non-ligand binding region
antibodies is done using a series of fusion proteins and deletions
of the membrane proximal region of the extracellular domain of a
human Notch1 from about amino acid 1427 to about amino acid 1732.
These recombinant fusion proteins are expressed in transiently
transfected HEK 293 cells from which conditioned medium is
collected twenty-four to forty-eight hours post-transfection for
ELISA.
In certain embodiments, the Notch1 fusion protein fragments are
separated on SDS-PAGE gels and probed with both anti-Fc antibodies
to detect the presence of all fusion proteins versus anti-Notch1
antibodies to detect the domains recognized by each anti-Notch
antibody.
To identify specific epitopes within the extracellular domains
recognized by an antibody against Notch1 the SPOTs system is used
(Sigma Genosys, The Woodlands, Tex.). A series of 10-residue linear
peptides overlapping by one amino acid and covering the entire
Notch1 extracellular domain are synthesized and covalently bound to
a cellulose membrane by the SPOT synthesis technique. The membrane
is preincubated for 8 hours at room temperature with blocking
buffer and hybridized with antibody overnight at 4.degree. C. The
membrane is then washed, incubated with a secondary antibody
conjugated to horseradish peroxidase (HRP) (Amersham Bioscience,
Piscataway, N.J.), re-washed, and visualized with signal
development solution containing 3-amino-9-ethylcarbazole. Specific
epitopes recognized by an antibody are thus determined.
Chimeric Antibodies
After monoclonal antibodies that specifically recognize a
non-ligand binding membrane proximal domain of the extracellular
domain of a Notch1 receptor are identified, these antibodies are
modified to overcome the human anti-mouse antibody (HAMA) immune
response when rodent antibodies are used as therapeutics agents.
The variable regions of the heavy-chain and light-chain of the
selected monoclonal antibody are isolated by RT-PCR from hybridoma
cells and ligated in-frame to human IgG1 heavy-chain and kappa
light chain constant regions, respectively, in mammalian expression
vectors. Alternatively a human Ig expression vector such as TCAE
5.3 is used that contains the human IgG1 heavy-chain and kappa
light-chain constant region genes on the same plasmid (Preston et
al., 1998, Infection & Immunity 66:4137-42). Expression vectors
encoding chimeric heavy- and light-chains are then co-transfected
into Chinese hamster ovary (CHO) cells for chimeric antibody
production. Immunoreactivity and affinity of chimeric antibodies
are compared to parental murine antibodies by ELISA and FACS.
Humanized Antibodies
As chimeric antibody therapeutics are still frequently antigenic,
producing a human anti-chimeric antibody (HACA) immune response,
chimeric antibodies against a non-ligand binding membrane proximal
domain of the extracellular domain of a Notch1 receptor can require
further humanization. To generate humanized antibodies the three
short hypervariable sequences, or complementary determining regions
(CDRs), of the chimeric antibody heavy- and light-chain variable
domains described above are engineered using recombinant DNA
technology into the variable domain framework of a human heavy- and
light-chain sequences, respectively, and then cloned into a
mammalian expression vector for expression in CHO cells. The
immunoreactivity and affinity of the humanized antibodies are
compared to parental chimeric antibodies by ELISA and FACS.
Additionally, site-directed or high-density mutagenesis of the
variable region can be used to optimize specificity, affinity, etc.
of the humanized antibody.
Example 2
Humanized antibodies against a membrane proximal region of the
extracellular domain of a human Notch1 were generated. The variable
domains of the murine monoclonal antibody 52M51 were isolated and
sequenced from the hybridoma line using degenerate PCR essentially
as described in Larrick, J. M., et al., 1989, Biochem. Biophys.
Res. Comm. 160: 1250 and Jones, S. T. & Bendig, M. M., 1991,
Bio/Technology 9: 88. Human heavy and light chain variable
framework regions likely to be structurally similar to the parental
52M51 antibody amino acid sequences are then considered as
reference human framework regions to help guide the design of novel
synthetic frameworks. To identify the human framework regions
bearing similarity to 52M51 murine frameworks, the predicted
protein sequences encoded by the V.sub.H and V.sub.L murine
variable domains of 52M51 are compared with human antibody
sequences encoded by expressed human cDNA using BLAST searches for
human sequence deposited in Genbank. Using this method, expressed
human cDNA sequences (e.g. genbank DA975021, DB242412) and germline
Vh domains (e.g. IGHV1-24) were selected for further analysis in
designing heavy chain frameworks. Similarly, expressed human cDNA
sequences (e.g. genbank CD709370, CD707373) and germline VI (e.g.
IGLV7-46, IGLV8-61) were considered in designing light chain
frameworks.
The amino acid differences between candidate humanized framework
heavy chains and the parent murine monoclonal antibody 52M51 heavy
chain variable domain and light chain variable domains were
evaluated for likely importance, and a judgment made as to whether
each difference in position contributes to proper folding and
function of the variable domain. This analysis was guided by
examination of solved crystal structures of other antibody
fragments (e.g., the structure of Fab 2E8 as described in Trakhanov
et al, Acta Crystallogr D Biol Crystallogr, 1999, 55:122-28, as
well as other protein crystal structures (e.g., protein data bank
structures 1ADQ and 1GIG)). Structures were modeled using computer
software including Jmol, quick PDB, and Pymol. Consideration was
given to the potential impact of an amino acid at a given position
on the packing of the .beta.-sheet framework, the interaction
between the heavy and light chain variable domains, the degree of
solvent exposure of the amino acid side chain, and the likelihood
that an amino acid would impact the positioning of the CDR loops.
From this analysis, nine candidate V.sub.H chains fused in-frame to
the human IgG2 constant region and eight candidate V1 chains fused
in frame with the human IgLC1 constant region were conceived and
chemically synthesized. The candidate heavy chains comprise: i) a
synthetic framework designed to resemble natural human frameworks
and ii) the parental 52M51 murine antibody CDRs.
The functionality of each candidate variant humanized heavy and
light chain was tested by cotransfection into mammalian cells. Each
of the nine candidate humanized 52M51 heavy chains described above
was cotransfected into HEK 293 cells with the murine 52M51 light
chain cDNA, and conditioned media was assayed by ELISA for Notch1
binding activity. The 52M51 heavy chain variant exhibiting the most
robust binding was selected. This variant "52M51-H4" (SEQ ID NO:22)
contains, in addition to murine CDRs, variation at 3 framework
positions within the Vh framework, Kabat positions 20, 48, and 71
in comparison with an example human framework (e.g. IGHV1-24). The
52M51-H4 humanized heavy chain was then cotransfected into HEK293
cells with each of the eight candidate humanized light chains, and
conditioned media was again assayed for antigen binding by ELISA.
Two light chain variants "2M51 L3" (SEQ ID NO:26) and "52M51 L4"
(SEQ ID NO:30) were found to exhibit better binding than the other
candidates and were chosen for further study. Variant 52M51-L3
contains, in addition to murine CDRs, variation at 1 framework
position at Kabat position 49 in comparison to an example human
framework (e.g., IGLV7-46). Two humanized variant antibodies,
52M51H4L3 and 52M51H4L4, were developed. 52M51H4L3, as encoded by
DNA deposited with the ATCC, under the conditions of the Budapest
Treaty on Oct. 15, 2008, and assigned designation number
PTA-9549.
The affinities for human and mouse Notch1 were determined using a
Biacore 2000 instrument. Briefly, recombinant human and mouse
Notch1 proteins were immobilized on a CM5 chip using standard amine
based chemistry (NHS/EDC). Different antibody concentrations were
injected over the protein surfaces and kinetic data were collected
over time. The data was fit using the simultaneous global fit
equation to yield dissociation constants (K.sub.D, nM) for each
Notch1 (Table 2).
TABLE-US-00002 TABLE 2 IgG Dissociation Constants (K.sub.D)
Antibody Human Notch1 (nM) Mouse Notch1 (nM) 52M51 2.86 NB
52M51H4L3 4.33 NB 52M51H4L4 7.35 NB
Example 3
Notch Receptor Signaling
In certain embodiments, the ability of Notch1 receptor antibodies
to block ligand-mediated Notch signaling was determined. In certain
embodiments, HeLa cells engineered to overexpress Notch1
(Notch1-Hela) cultured in DMEM supplemented with antibiotics and
10% FCS were co-transfected with 1) pGL4 8.times.CBS firefly
luciferase containing a Notch responsive promoter upstream of a
firefly luciferase reporter gene to measure Notch signaling levels
in response to DLL4 ligand; and 2) a Renilla luciferase reporter
(Promega; Madison, Wis.) as an internal control for transfection
efficiency. Transfected cells were added to cultures plates coated
overnight with 200 ng/well of hDLL4-fc protein, and antibodies to
Notch1 were then added to the cell culture medium. Forty-eight
hours following transfection, luciferase levels were measured using
a dual luciferase assay kit (Promega; Madison, Wis.) with firefly
luciferase activity normalized to Renilla luciferase activity. The
ability of antibodies to inhibit Notch1 pathway activation was thus
determined. Antibodies 52M51, 52M63, 52M74, and 52M80, generated
against a membrane proximal region of the extracellular domain of a
human Notch1 (FIG. 1A) significantly reduced luciferase activity
indicative of reduced Notch1 signaling as compared to other Notch1
antibodies (FIG. 1B). Further, a humanized variant of antibody
52M51, variant 52M51 H4/L3 displayed similar potency in reducing
luciferase activity (FIG. 1C).
Notch Receptor Activation and ICD Formation
Cleavage of Notch receptors by furin, ADAM, and gamma-secretase
results in formation of the Notch intracellular domain (ICD) that
then triggers downstream Notch signaling in the nucleus. In certain
embodiments, the ability of Notch1 receptor antibodies to block
ligand-mediated receptor activation was determined by Western blot
analysis. Notch1-Hela cells were grown in suspension culture in
293-SMII media (Gibco). Cultured cells were transferred to 96-well
plates in which select wells had been pre-coated with human DLL4-fc
fusion protein (2 .mu.g/ml) in DMEM plus 2% FBS and 1 .mu.M MG132
(Calbiochem). Antibodies to generated against a membrane proximal
region of the extracellular domain of human Notch1 were added to
the cell culture medium, and cells were incubated at 37.degree. C.
for five hours. Wells were then aspirated and the cells resuspended
in 2.times.SDS running buffer. Samples were sonicated at room
temperature, and then subjected to SDS-PAGE and western blot
analysis using an antibody specific for the cleaved Notch1 ICD
according to the manufacturer's recommendations (Cell Signaling
Technology). 52M51 along with 52M63, 52M74, and 52M80 all
significantly inhibited the generation of ICD after ligand
stimulation (FIG. 1D).
Example 4
In vivo Prevention of Tumor Growth Using Non-Ligand Binding Region
Anti-Notch Receptor Antibodies
Tumor cells from a patient sample that have been passaged as a
xenograft in mice were prepared for injection into experimental
animals. Tumors were established at OncoMed Pharmaceuticals by
adhering to procedures described previously (See Al-Hajj et al.,
2003; Dalerba et al., 2007) and include: UM-PE13 and T3 (breast
carcinoma cells), OMP-C9, OMP-C8, OMP-C6, and Colo-205 (colon tumor
cells); and OMP-PN4 (pancreatic carcinoma cells). Tumor tissue was
removed under sterile conditions, cut up into small pieces, minced
completely using sterile blades, and single cell suspensions
obtained by enzymatic digestion and mechanical disruption. The
resulting tumor pieces were mixed with ultra-pure collagenase III
in culture medium (200-250 units of collagenase per mL) and
incubated at 37.degree. C. for 3-4 hours with pipetting up and down
through a 10-mL pipette every 15-20 min. Digested cells were
filtered through a 45 ul nylon mesh, washed with RPMI/20% FBS, and
washed twice with HBSS. Dissociated tumor cells were then injected
subcutaneously into NOD/SCID mice at 6-8 weeks to elicit tumor
growth. For UM-PE13 and T3 breast tumor cells, 50,000 cells in 100
ul were injected into the right mammary fat pad (n=20) along with
the implantation of an estrogen pellet. For OMP-C9 colon tumor
cells, 50,000 cells in 100 ul were injected into the right flank
region (n=20). For OMP-C8 colon tumor cells, 10,000 cells in 100 ul
were injected into the right flank area (n=10). For OMP-C6 colon
tumor cells, 10,000 cells in 100 ul were injected into the right
flank (n=10). All tumor cells were injected in a mixture of PBS
(without magnesium or calcium) and BD Matrigel (BD Biosciences) at
a 1:1 ratio.
Three days after tumor cell injection, antibody treatment was
commenced. Each injected animal received 10 mg/kg anti-Notch1
antibodies or PBS as a control intraperitoneal (i.p.) two times per
week for a total of 6 to 8 weeks. Animals injected with PE13 cells
received injections into the right upper mammary fat pad in
addition to estrogen pellet injections. Animals injected with C9,
C8, or C6 cells received injections in the right lower quadrant of
the abdomen. Tumor size was assessed twice a week.
In certain embodiments, antibodies against a membrane proximal
region of the extracellular domain of human Notch1 were tested for
an effect on the formation of breast tumors. PE13 breast tumor
cells (50,000 cells per injection) were implanted subcutaneously
into the mammary fat pads. Two days following cell implantation,
animals were treated with either control antibody or 52M antibodies
52M1, 52M2, and 52M8 (which were without anti-Notch signaling
capability, see FIG. 1B) at 10 mg/kg dosed i.p. twice a week.
Treatment with non-Notch1 inhibitor antibodies had no effect on
tumor growth compared to control treated animals (FIGS. 2C and 2D).
In certain embodiments, animals injected with PE13 breast tumor
cells are treated with either control antibody or 52M51 at 10 mg/kg
dosed i.p. twice a week. Tumor volume is measured twice weekly, and
the effect of 52M51 on breast tumor growth is determined.
In alternative embodiments, dissociated tumor cells are first
sorted into tumorigenic and non-tumorigenic cells based on cell
surface markers before injection into experimental animals.
Specifically, tumor cells dissociated as described above are washed
twice with Hepes buffered saline solution (HBSS) containing 2%
heat-inactivated calf serum (HICS) and resuspended at 10.sup.6
cells per 100 ul. Antibodies are added and the cells incubated for
20 min on ice followed by two washes with HBSS/2% HICS. Antibodies
include anti-ESA (Biomeda, Foster City, Calif.), anti-CD44,
anti-CD24, and Lineage markers anti-CD2, -CD3, -CD10, -CD16, -CD18,
-CD31, -CD64, and -CD140b (collectively referred to as Lin;
PharMingen, San Jose, Calif.). Antibodies are directly conjugated
to fluorochromes to positively or negatively select cells
expressing these markers. Mouse cells are eliminated by selecting
against H2Kd+ cells, and dead cells are eliminated by using the
viability dye 7AAD. Flow cytometry is performed on a FACSVantage
(Becton Dickinson, Franklin Lakes, N.J.). Side scatter and forward
scatter profiles are used to eliminate cell clumps. Isolated ESA+,
CD44+, CD24-/low, Lin-tumorigenic cells are then injected
subcutaneously into NOD/SCID mice to elicit tumor growth.
Example 5
In vivo Treatment of Tumors Using Anti-Notch1 Receptor
Antibodies
Tumor cells from a patient sample (solid tumor biopsy or pleural
effusion) that have been passaged as a xenograft in mice were
prepared for repassaging into experimental animals. Tumor tissue
was removed, cut up into small pieces, minced completely using
sterile blades, and single cell suspensions obtained by enzymatic
digestion and mechanical disruption. Dissociated tumor cells were
then injected subcutaneously into the mammary fat pads, for breast
tumors, or into the flank, for non-breast tumors, of NOD/SCID mice
to elicit tumor growth. In certain embodiments, ESA+, CD44+,
CD24-/low, Lin-tumorigenic tumor cells are isolated as described in
detail above and injected.
In certain embodiments, freshly isolated C8 colon tumor cells (225
cells per animal) were implanted subcutaneously into NOD/SCID mice.
Following tumor cell injection, animals were monitored for tumor
growth. Tumors were allowed to grow for 48 days until they reached
an average size of approximately 210 mm.sup.3 and randomized into
two groups (n=10 per group). The animals were treated with either
control antibody or antibody that binds to the membrane proximal
region of the extracellular domain of human Notch1, 52M51, (10
mg/kg) dosed i.p. twice a week. Tumor size was assessed on days 55,
57, and 62. Animals treated with 52M51 showed a statistically
significant (p=0.0006) inhibition of tumor growth compared to
control treated animals (FIGS. 2A and 2B).
At the end point of antibody treatment, tumors are harvested for
further analysis. In some embodiments, a portion of the tumor is
analyzed by immunofluorescence to assess antibody penetration into
the tumor and tumor response. A portion of each harvested tumor
from anti-Notch1 receptor treated and control antibody treated mice
is flash-frozen in liquid nitrogen, embedded in O.C.T., and cut on
a cryostat as 10 um sections onto glass slides. Alternatively a
portion of each tumor is formalin-fixed, paraffin-embedded, and cut
on a microtome as 10 um section onto glass slides. Sections are
post-fixed and incubated with chromophore labeled antibodies that
specifically recognize injected antibodies to detect anti-NOTCH1
receptor or control antibodies present in the tumor biopsy.
Furthermore antibodies that detect different tumor and tumor
recruited cell types such as, for example, anti-VE cadherin (CD144)
or anti-PECAM-1 (CD31) antibodies to detect vascular endothelial
cells, anti-smooth muscle alpha-actin antibodies detect vascular
smooth muscle cells, anti-Ki67 antibodies to detect proliferating
cells, TUNEL assays to detect dying cells, and anti-intracellular
domain (ICD) Notch fragment antibodies to detect Notch signaling
can be used to assess affects of antibody treatment on
angiogenesis, tumor growth and tumor morphology.
The effect of anti-Notch1 receptor antibody treatment on tumor cell
gene expression is also assessed. Total RNA is extracted from a
portion of each harvested tumor from Notch1 antibody treated and
control antibody treated mice and used for quantitative RT-PCR.
Expression levels of Notch1, components of Notch signaling pathway
including, as well as addition cancer stem cell markers previously
identified including, for example, CD44 are analyzed relative to
the house-keeping gene GAPDH as an internal control. Changes in
tumor cell gene expression upon Notch1 receptor antibody treatment
are thus determined.
In addition, the effect of anti-Notch1 receptor antibody treatment
on the presence of cancer stem cells in a tumor is assessed. Tumor
samples from Notch1 versus control antibody treated mice are cut up
into small pieces, minced completely using sterile blades, and
single cell suspensions obtained by enzymatic digestion and
mechanical disruption. Dissociated tumor cells are then analyzed by
FACS analysis for the presence of tumorigenic cancer stem cells
based on ESA+, CD44+, CD24-/low, Lin-surface cell marker expression
as described in detail above.
The tumorigenicity of cells isolated based on ESA+, CD44+,
CD24-/low, Lin-expression following anti-Notch1 antibody treatment
can then be assessed. 5,000, 1,000, 500, and 100 isolated ESA+,
CD44+, CD24-/low, Lin-cancer stem cells from Notch1 antibody
treated versus control antibody treated mice are re-injected
subcutaneously into the mammary fat pads of NOD/SCID mice. The
tumorigenicity of cancer stem cells based on the number of injected
cells required for consistent tumor formation is thus
determined.
In contrast to the in vivo efficacy of 52M51, an antibody that
inhibits Notch1 signaling, in a colon xenograft model described
above, certain other antibodies that recognize the membrane
proximal region of Notch 1, but don't inhibit Notch 1 signaling,
were found to not have anti-tumor efficacy in vivo in a breast
xenograft model. The antibodies 52M51, 52M2, and 52M8, each of
which had been found to not appreciably inhibit Notch signaling
(Example 3 and FIG. 1B), were injected in NOD/SCID mice which had
been previously injected with PE13 breast tumor cells. Each of the
antibodies 52M1, 52M2, and 52M8 failed to effect tumor growth in
the xenograft model when compared against control-treated animals
(FIG. 2C (52M1, 52M2) and FIG. 2D (52M8)).
Example 6
Treatment of Human Cancer Using Anti-Notch Receptor Antibodies
This example describes methods for treating cancer using antibodies
against a Notch receptor to target tumors comprising cancer stem
cells and/or tumor cells in which Notch receptor expression has
been detected.
The presence of cancer stem cell marker expression can first be
determined from a tumor biopsy. Tumor cells from a biopsy from a
patient diagnosed with cancer are removed under sterile conditions.
In some embodiments, the tissue biopsy is fresh-frozen in liquid
nitrogen, embedded in O.C.T., and cut on a cryostat as 10 um
sections onto glass slides. Alternatively the tissue biopsy is
formalin-fixed, paraffin-embedded, and cut on a microtome as 10 um
section onto glass slides. Sections are incubated with antibodies
against a Notch receptor to detect protein expression.
Additionally, the presence of cancer stem cells can be determined.
Tissue biopsy samples are cut up into small pieces, minced
completely using sterile blades, and cells subject to enzymatic
digestion and mechanical disruption to obtain a single cell
suspension. Dissociated tumor cells are then incubated with
anti-ESA, -CD44, -CD24, -Lin, and -Notch1 antibodies to detect
cancer stem cells, and the presence of ESA+, CD44+, CD24-/low,
Lin-, Notch+ tumor stem cells is determined by flow cytometry as
described in detail above.
Cancer patients whose tumors are diagnosed as expressing a Notch
receptor are treated with anti-Notch receptor antibodies. Humanized
or human monoclonal anti-Notch receptor antibodies generated as
described above are purified and formulated with a suitable
pharmaceutical carrier in PBS for injection. Patients are treated
with the Notch antibodies once a week for at least 10 weeks, but in
certain cases once a week for at least about 14 weeks. Each
administration of the antibody should be a pharmaceutically
effective dose about 2 to about 100 mg/ml and in certain cases
between about 5 to about 40 mg/ml. The antibody can be administered
prior to, concurrently with, or after standard radiotherapy
regimens or chemotherapy regimens using one or more
chemotherapeutic agent, such as oxaliplatin, fluorouracil,
leucovorin, or streptozocin. Patients are monitored to determine
whether such treatment has resulted in an anti-tumor response, for
example, based on tumor regression, reduction in the incidences of
new tumors, lower tumor antigen expression, decreased numbers of
cancer stem cells, or other means of evaluating disease
prognosis.
Example 7
Additional Studies of in vivo Treatment of Tumors Using Anti-Notch1
Receptor Antibodies
In one embodiment, M2 melanoma cells (10,000) were injected
subcutaneously in NOD-SCID mice. Tumors were allowed to grow for 35
days until they had reached a volume of approximately 110 mm.sup.3.
Tumor-bearing mice were randomized into two groups (n=10) and
treated with either control antibody or anti-Notch1 antibody 52R43.
Antibodies were dosed twice weekly at 10 mg/kg. Tumor volumes were
measured on the indicated days. As shown in FIG. 3A, anti-Notch1
treatment with 52R43 reduced tumor growth relative to the control
group (p=0.02).
In one embodiment, Lu24 lung tumor cells (30,000) were injected
subcutaneously in NOD-SCID mice. Tumors were allowed to grow for 35
days until they had reached a volume of approximately 205 mm.sup.3.
Tumor-bearing mice were randomized into two groups (n=8) and
treated with either control antibody or anti-Notch1 antibody 52R43.
Antibodies were dosed twice weekly at 10 mg/kg. Tumor volumes were
measured on the indicated days. As shown in FIG. 3B, anti-Notch1
treatment with 52R43 reduced tumor growth relative to the control
group (p=0.04).
In one embodiment, PN8 pancreatic tumor cells (50,000) were
injected subcutaneously in NOD-SCID mice. Tumors were allowed to
grow for 27 days until they had reached a volume of approximately
115 mm.sup.3. Tumor bearing mice were randomized into two groups
(n=8) and treated with either control antibody or anti-Notch1
antibody 52R43. Antibodies were dosed twice weekly at 10 mg/kg.
Tumor volumes were measured on the indicated days. As shown in FIG.
3C, anti-Notch1 treatment with 52R43 reduced tumor growth relative
to the control group (p=0.005).
In one embodiment, T1 breast tumor cells (300,000) were injected
subcutaneously in NOD-SCID mice. Tumors were allowed to grow for 27
days until they had reached a volume of approximately 130 mm.sup.3.
Tumor bearing mice were randomized into four groups (n=10) and
treated with either control antibody, anti-Notch1 52R43, taxol, or
a combination of 52R43 and taxol. Antibodies were dosed once weekly
at 15 mg/kg and taxol was dosed once weekly at 12 mg/kg. Tumor
volumes were measured on the indicated days. As shown in FIG. 3D,
anti-Notch1 treatment with 52R43 reduced tumor growth relative to
the control group (p<0.0001), and the combination group was
reduced relative to taxol alone (p=0.001)
All publications and patents mentioned in the above specification
are herein incorporated by reference. Various modifications and
variations of the described method and system of the invention will
be apparent to those in the art without departing from the scope
and spirit of the invention. Although the invention has been
described in connection with specific embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those in the relevant fields are intended to be within
the scope of the following claims.
TABLE-US-00003 SEQUENCES SEQ ID NO: 1 Notch1 Polynucleotide
encoding amino acids 1427-1732.
CACATCCTGGACTACAGCTTCGGGGGTGGGGCCGGGCGCGACATCCCCCCGCCGCTGATC
GAGGAGGCGTGCGAGCTGCCCGAGTGCCAGGAGGACGCGGGCAACAAGGTCTGCAGCCTG
CAGTGCAACAACCACGCGTGCGGCTGGGACGGCGGTGACTGCTCCCTCAACTTCAATGAC
CCCTGGAAGAACTGCACGCAGTCTCTGCAGTGCTGGAAGTACTTCAGTGACGGCCACTGT
GACAGCCAGTGCAACTCAGCCGGCTGCCTCTTCGACGGCTTTGACTGCCAGCGTGCGGAA
GGCCAGTGCAACCCCCTGTACGACCAGTACTGCAAGGACCACTTCAGCGACGGGCACTGC
GACCAGGGCTGCAACAGCGCGGAGTGCGAGTGGGACGGGCTGGACTGTGCGGAGCATGTA
CCCGAGAGGCTGGCGGCCGGCACGCTGGTGGTGGTGGTGCTGATGCCGCCGGAGCAGCTG
CGCAACAGCTCCTTCCACTTCCTGCGGGAGCTCAGCCGCGTGCTGCACACCAACGTGGTC
TTCAAGCGTGACGCACACGGCCAGCAGATGATCTTCCCCTACTACGGCCGCGAGGAGGAG
CTGCGCAAGCACCCCATCAAGCGTGCCGCCGAGGGCTGGGCCGCACCTGACGCCCTGCTG
GGCCAGGTGAAGGCCTCGCTGCTCCCTGGTGGCAGCGAGGGTGGGCGGCGGCGGAGGGAG
CTGGACCCCATGGACGTCCGCGGCTCCATCGTCTACCTGGAGATTGACAACCGGCAGTGT
GTGCAGGCCTCCTCGCAGTGCTTCCAGAGTGCCACCGACGTGGCCGCATTCCTGGGAGCG
CTCGCCTCGCTGGGCAGCCTCAACATCCCCTACAAGATCGAGGCCGTGCAGAGTGAGACC
GTGGAGCCGCCCCCGCCG SEQ ID NO: 2 Notch1 amino acids 1427-1732
HILDYSFGGGAGRDIPPPLIEEACELPECQEDAGNKVCSLQCNNHACGWDGGDCSLNFND
PWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQRAEGQCNPLYDQYCKDHFSDGHC
DQGCNSAECEWDGLDCAEHVPERLAAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNVV
FKRDAHGQQMIFPYYGREEELRKHPIKRAAEGWAAPDALLGQVKASLLPGGSEGGRRRRE
LDPMDVRGSIVYLEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLNIPYKIEAVQSET VEPPPP
Mouse antibody 52M51 sequences: SEQ ID NO: 3 52M51 Light chain
polynucleotide sequence (Putative signal sequence is underlined)
ATGGCCTGGATTTCACTTATACTCTCTCTCCTGGCTCTCAGCTCAGGGGCCATTTCCCAG
GCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACT
TGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTACGCCAACTGGGTCCAAGAAAAA
CCTGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCT
GCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAG
ACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTCGGT
GGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTT
CCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGAT
TTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGT
ATGGAGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTG
ACCCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAA
GGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCCTAG SEQ ID NO: 4 52M51
Light chain amino acid sequence (Putative signal sequence is
underlined)
MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEK
PDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFG
GGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQG
METTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS SEQ ID NO: 5
52M51 Light chain variable region polynucleotide sequence (Putative
signal sequence is underlined)
ATGGCCTGGATTTCACTTATACTCTCTCTCCTGGCTCTCAGCTCAGGGGCCATTTCCCAG
GCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACT
TGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTACGCCAACTGGGTCCAAGAAAAA
CCTGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCT
GCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAG
ACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTCGGT
GGAGGAACCAAACTGACTGTCCTAGGC SEQ ID NO: 6 52M51 Light chain variable
region amino acid sequence (Putative signal sequence is underlined)
MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEK
PDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFG
GGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQG SEQ ID
NO: 7 52M51 Light chain variable region polynucleotide sequence
without putative signal sequence
CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTC
ACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTACGCCAACTGGGTCCAAGAA
AAACCTGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTT
CCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCA
CAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTC
GGTGGAGGAACCAAACTGACTGTCCTAGGC SEQ ID NO: 8 52M51 Light chain
variable region amino acid sequence without putative signal
sequence
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGV
PARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLG SEQ ID NO: 9
52M51 Heavy chain polynucleotide sequence (Putative signal sequence
is underlined)
ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAG
GTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCC
TGCAAGGCTGCTGGCTACACAATGAGAGGCTACTGGATAGAGTGGATAAAGCAGAGGCCT
GGACATGGCCTTGAGTGGATTGGACAGATTTTACCTGGAACTGGGAGAACTAACTACAAT
GAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCAACATG
CAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATTTGATGGT
AACTACGGTTACTATGCTATGGACTACTGGGGTCAAGGATCCTCAGTCACCGTCTCCTCA
GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAAC
TCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACC
TGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGAC
CTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTC
ACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG
GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTC
CCCCCAAAGCCCAAGGATGTCCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTG
GTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAG
GTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTC
AGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTC
AACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATATCCAAAACCAAAGGCAGACCG
AAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTC
AGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATAACAGTGGAGTGGCAGTGG
AATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGAACACGAATGGCTCT
TACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTC
ACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCAC
TCTCCTGGTAAATGA SEQ ID NO: 10 52M51 Heavy chain amino acid sequence
(Putative signal sequence is underlined)
MEWTWVFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKISCKAAGYTMRGYWIEWIKQRP
GHGLEWIGQILPGTGRTNYNEKFKGKATFTADTSSNTANMQLSSLTSEDSAVYYCARFDG
NYGYYAMDYWGQGSSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVT
WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPR
DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVE
VHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRP
KAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGS
YFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 11 52M51
Heavy chain variable region polynucleotide sequence (Putative
signal sequence is underlined)
ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAG
GTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCC
TGCAAGGCTGCTGGCTACACAATGAGAGGCTACTGGATAGAGTGGATAAAGCAGAGGCCT
GGACATGGCCTTGAGTGGATTGGACAGATTTTACCTGGAACTGGGAGAACTAACTACAAT
GAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCAACATG
CAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATTTGATGGT
AACTACGGTTACTATGCTATGGACTACTGGGGTCAAGGATCCTCAGTCACCGTCTCCTCA SEQ ID
NO: 12 52M51 Heavy chain variable region amino acid sequence
(Putative signal sequence is underlined)
MEWTWVFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKISCKAAGYTMRGYWIEWIKQRP
GHGLEWIGQILPGTGRTNYNEKFKGKATFTADTSSNTANMQLSSLTSEDSAVYYCARFDG
NYGYYAMDYWGQGSSVTVSSAKTTPPSVYPLAPGSAAQTNVTLGCLVKGYFPEPVTVT SEQ ID
NO: 13 52M51 Heavy chain variable region polynucleotide sequence
without putative signal sequence
CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATA
TCCTGCAAGGCTGCTGGCTACACAATGAGAGGCTACTGGATAGAGTGGATAAAGCAGAGG
CCTGGACATGGCCTTGAGTGGATTGGACAGATTTTACCTGGAACTGGGAGAACTAACTAC
AATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCAAC
ATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATTTGAT
GGTAACTACGGTTACTATGCTATGGACTACTGGGGTCAAGGATCCTCAGTCACCGTCTCC TCA
SEQ ID NO: 14 52M51 Heavy chain variable region amino acid sequence
without putative signal sequence
QVQLQQSGAELMKPGASVKISCKAAGYTMRGYWIEWIKQRPGHGLEWIGQILPGTGRTNY
NEKFKGKATFTADTSSNTANMQLSSLTSEDSAVYYCARFDGNYGYYAMDYWGQGSSVTVS SA SEQ
ID NO: 15 52M51 Heavy chain CDR1 RGYWIE SEQ ID NO: 16 52M51 Heavy
chain CDR2 QILPGTGRTNYNEKFKG SEQ ID NO: 17 52M51 Heavy chain CDR3
FDGNYGYYAMDY SEQ ID NO: 18 52M51 Light chain CDR1 RSSTGAVTTSNYAN
SEQ ID NO: 19 52M51 Light chain CDR2 GTNNRAP SEQ ID NO: 20 52M51
Light chain CDR3 ALWYSNHWVFGGGTKL Humanized 52M51 sequences: SEQ ID
NO: 21 52M51-H4 Heavy chain polynucleotide sequence (Putative
signal sequence underlined)
ATGGATTGGACATGGAGGGTGTTCTGCCTCCTCGCTGTGGCTCCTGGAGTCCTGAGCCAG
GTCCAGCTCGTCCAGAGCGGGGCTGAAGTCAAGAAGCCTGGCGCTAGCGTCAAAATCAGC
TGTAAGGTCAGCGGATACACACTGAGGGGATACTGGATCGAGTGGGTGAGGCAGGCTCCA
GGAAAGGGCCTGGAATGGATCGGCCAGATCCTGCCTGGAACCGGAAGGACAAATTACAAT
GAGAAGTTTAAGGGAAGGGTCACAATGACAGCAGACACAAGCACAGACACAGCTTATATG
GAACTCAGCTCCCTCAGATCCGAGGACACCGCTGTCTACTATTGTGCCAGGTTCGATGGA
AATTACGGATACTATGCCATGGATTACTGGGGACAGGGGACAACGGTCACCGTGAGCTCA
GCCAGCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACCAGCGAG
AGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCA
GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACC
TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGC
AAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTC
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGC
GTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGC
GTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGT
GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGG
CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAATGA SEQ ID NO: 22 52M51 H4 Heavy chain amino acid
sequence (Putative signal sequence underlined)
MDWTWRVFCLLAVAPGVLSQVQLVQSGAEVKKPGASVKISCKVSGYTLRGYWIEWVRQAP
GKGLEWIGQILPGTGRTNYNEKFKGRVTMTADTSTDTAYMELSSLRSEDTAVYYCARFDG
NYGYYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER
KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG
VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 23
52M51-H4 Heavy chain variable region amino acid sequence (Putative
signal sequence underlined)
MDWTWRVFCLLAVAPGVLSQVQLVQSGAEVKKPGASVKISCKVSGYTLRGYWIEWVRQAP
GKGLEWIGQILPGTGRTNYNEKFKGRVTMTADTSTDTAYMELSSLRSEDTAVYYCARFDG
NYGYYAMDYWGQGTTVTVSSA SEQ ID NO: 24 52M51-H4 Heavy chain variable
region amino acid sequence without putative signal sequence
QVQLVQSGAEVKKPGASVKISCKVSGYTLRGYWIEWVRQAPGKGLEWIGQILPGTGRTNY
NEKFKGRVTMTADTSTDTAYMELSSLRSEDTAVYYCARFDGNYGYYAMDYWGQGTTVTVS SA SEQ
ID NO: 25 52M51-L3 Light chain polynucleotide sequence (Putative
signal sequence is underlined)
ATGAGCGTCCCTACAATGGCTTGGATGATGCTCCTGCTGGGACTCCTGGCTTATGGAAGC
GGAGTGGATAGCCAGGCCGTCGTCACACAGGAACCTAGCCTCACCGTTAGCCCTGGAGGA
ACAGTCACACTGACCTGTAGGAGCTCCACAGGAGCTGTGACAACAAGCAATTACGCTAAC
TGGTTCCAGCAGAAGCCCGGTCAAGCCCCTAGAACCCTCATCGGCGGCACCAATAACAGA
GCTCCCGGAGTCCCCGCCAGGTTCTCCGGCTCCCTCCTGGGTGGCAAGGCTGCTCTGACA
CTCAGCGGTGCCCAGCCAGAGGATGAAGCGGAGTACTACTGTGCACTGTGGTACAGCAAC
CATTGGGTTTTCGGAGGCGGAACAAAGTTAACCGTCCTCGGGCAGCCTAAGGCTGCTCCT
AGCGTCACACTGTTCCCCCCATCTAGCGAGGAGCTGCAGGCTAACAAGGCAACCCTCGTC
TGCCTGGTTAGCGACTTCTACCCTGGCGCTGTCACAGTGGCCTGGAAAGCTGACGGCTCC
CCTGTGAAAGTTGGCGTCGAAACCACAAAGCCTTCTAAGCAGAGCAATAATAAATATGCC
GCAAGCTCCTACCTCTCCCTGACTCCTGAGCAGTGGAAAAGCCATAGGAGCTACTCCTGC
CGGGTCACACACGAAGGAAGCACAGTGGAAAAGACAGTCGCCCCTGCTGAGTGTAGCTGA SEQ ID
NO: 26 52M51-L3 Light chain amino acid sequence (Putative signal
sequence is underlined)
MSVPTMAWMMLLLGLLAYGSGVDSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYAN
WFQQKPGQAPRTLIGGTNNRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSN
HWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
PVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS SEQ ID
NO: 27 52M51-L3 Light chain variable region amino acid sequence
(Putative signal sequence is underlined)
MSVPTMAWMMLLLGLLAYGSGVDSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYAN
WFQQKPGQAPRTLIGGTNNRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSN
HWVFGGGTKLTVLG SEQ ID NO: 28 52M51-L3 Light chain variable region
amino acid sequence without putative signal sequence
SGVDSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRTLIGGTNN
RAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLG SEQ ID NO:
29 52M51-L4 Light chain polynucleotide sequence (Putative signal
sequence is underlined)
ATGAGCGTCCCTACAATGGCTTGGATGATGCTCCTGCTGGGACTCCTGGCTTATGGAAGC
GGAGTGGATAGCCAGACCGTCGTCACACAGGAACCTAGCTTTTCCGTTAGCCCTGGAGGA
ACAGTCACACTGACCTGTAGGAGCTCCACAGGAGCTGTGACAACAAGCAATTACGCTAAC
TGGTATCAGCAGACTCCCGGTCAAGCCCCTAGAACCCTCATCGGCGGCACCAATAACAGA
GCTCCCGGAGTCCCCGACAGGTTCTCCGGCTCCATCCTGGGAAATAAAGCTGCTCTGACA
ATCACAGGTGCCCAGGCTGACGATGAAAGCGACTACTACTGTGCACTGTGGTACAGCAAC
CATTGGGTTTTCGGAGGCGGAACAAAGTTAACCGTCCTCGGGCAGCCTAAGGCTGCTCCT
AGCGTCACACTGTTCCCCCCATCTAGCGAGGAGCTGCAGGCTAACAAGGCAACCCTCGTC
TGCCTGGTTAGCGACTTCTACCCTGGCGCTGTCACAGTGGCCTGGAAAGCTGACGGCTCC
CCTGTGAAAGTTGGCGTCGAAACCACAAAGCCTTCTAAGCAGAGCAATAATAAATATGCC
GCAAGCTCCTACCTCTCCCTGACTCCTGAGCAGTGGAAAAGCCATAGGAGCTACTCCTGC
CGGGTCACACACGAAGGAAGCACAGTGGAAAAGACAGTCGCCCCTGCTGAGTGTAGCTGA SEQ ID
NO: 30 52M51-L4 Light chain amino acid sequence (Putative signal
sequence is underlined)
MSVPTMAWMMLLLGLLAYGSGVDSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYAN
WYQQTPGQAPRTLIGGTNNRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSN
HWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGS
PVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS SEQ ID
NO: 31 52M51-L4 Light chain variable region amino acid sequence
(Putative signal sequence is underlined)
MSVPTMAWMMLLLGLLAYGSGVDSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYAN
WYQQTPGQAPRTLIGGTNNRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSN
HWVFGGGTKLTVLG SEQ ID NO: 32 52M51-L4 Light chain variable region
amino acid sequence without putative signal sequence
SGVDSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWYQQTPGQAPRTLIGGTNN
RAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNHWVFGGGTKLTVLG
SEQUENCE LISTINGS
1
321918DNAHomo sapiens 1cacatcctgg actacagctt cgggggtggg gccgggcgcg
acatcccccc gccgctgatc 60gaggaggcgt gcgagctgcc cgagtgccag gaggacgcgg
gcaacaaggt ctgcagcctg 120cagtgcaaca accacgcgtg cggctgggac
ggcggtgact gctccctcaa cttcaatgac 180ccctggaaga actgcacgca
gtctctgcag tgctggaagt acttcagtga cggccactgt 240gacagccagt
gcaactcagc cggctgcctc ttcgacggct ttgactgcca gcgtgcggaa
300ggccagtgca accccctgta cgaccagtac tgcaaggacc acttcagcga
cgggcactgc 360gaccagggct gcaacagcgc ggagtgcgag tgggacgggc
tggactgtgc ggagcatgta 420cccgagaggc tggcggccgg cacgctggtg
gtggtggtgc tgatgccgcc ggagcagctg 480cgcaacagct ccttccactt
cctgcgggag ctcagccgcg tgctgcacac caacgtggtc 540ttcaagcgtg
acgcacacgg ccagcagatg atcttcccct actacggccg cgaggaggag
600ctgcgcaagc accccatcaa gcgtgccgcc gagggctggg ccgcacctga
cgccctgctg 660ggccaggtga aggcctcgct gctccctggt ggcagcgagg
gtgggcggcg gcggagggag 720ctggacccca tggacgtccg cggctccatc
gtctacctgg agattgacaa ccggcagtgt 780gtgcaggcct cctcgcagtg
cttccagagt gccaccgacg tggccgcatt cctgggagcg 840ctcgcctcgc
tgggcagcct caacatcccc tacaagatcg aggccgtgca gagtgagacc
900gtggagccgc ccccgccg 9182306PRTHomo sapiens 2His Ile Leu Asp Tyr
Ser Phe Gly Gly Gly Ala Gly Arg Asp Ile Pro1 5 10 15Pro Pro Leu Ile
Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Glu Asp 20 25 30Ala Gly Asn
Lys Val Cys Ser Leu Gln Cys Asn Asn His Ala Cys Gly 35 40 45Trp Asp
Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 50 55 60Cys
Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys65 70 75
80Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys
85 90 95Gln Arg Ala Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys
Lys 100 105 110Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn
Ser Ala Glu 115 120 125Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His
Val Pro Glu Arg Leu 130 135 140Ala Ala Gly Thr Leu Val Val Val Val
Leu Met Pro Pro Glu Gln Leu145 150 155 160Arg Asn Ser Ser Phe His
Phe Leu Arg Glu Leu Ser Arg Val Leu His 165 170 175Thr Asn Val Val
Phe Lys Arg Asp Ala His Gly Gln Gln Met Ile Phe 180 185 190Pro Tyr
Tyr Gly Arg Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg 195 200
205Ala Ala Glu Gly Trp Ala Ala Pro Asp Ala Leu Leu Gly Gln Val Lys
210 215 220Ala Ser Leu Leu Pro Gly Gly Ser Glu Gly Gly Arg Arg Arg
Arg Glu225 230 235 240Leu Asp Pro Met Asp Val Arg Gly Ser Ile Val
Tyr Leu Glu Ile Asp 245 250 255Asn Arg Gln Cys Val Gln Ala Ser Ser
Gln Cys Phe Gln Ser Ala Thr 260 265 270Asp Val Ala Ala Phe Leu Gly
Ala Leu Ala Ser Leu Gly Ser Leu Asn 275 280 285Ile Pro Tyr Lys Ile
Glu Ala Val Gln Ser Glu Thr Val Glu Pro Pro 290 295 300Pro
Pro3053705DNAArtificial SequenceSynthetic light chain of antibody
52M51 3atggcctgga tttcacttat actctctctc ctggctctca gctcaggggc
catttcccag 60gctgttgtga ctcaggaatc tgcactcacc acatcacctg gtgaaacagt
cacactcact 120tgtcgctcaa gtactggggc tgttacaact agtaactacg
ccaactgggt ccaagaaaaa 180cctgatcatt tattcactgg tctaataggt
ggtaccaaca accgagctcc aggtgttcct 240gccagattct caggctccct
gattggagac aaggctgccc tcaccatcac aggggcacag 300actgaggatg
aggcaatata tttctgtgct ctatggtaca gcaaccactg ggtgttcggt
360ggaggaacca aactgactgt cctaggccag cccaagtctt cgccatcagt
caccctgttt 420ccaccttcct ctgaagagct cgagactaac aaggccacac
tggtgtgtac gatcactgat 480ttctacccag gtgtggtgac agtggactgg
aaggtagatg gtacccctgt cactcagggt 540atggagacaa cccagccttc
caaacagagc aacaacaagt acatggctag cagctacctg 600accctgacag
caagagcatg ggaaaggcat agcagttaca gctgccaggt cactcatgaa
660ggtcacactg tggagaagag tttgtcccgt gctgactgtt cctag
7054234PRTArtificial SequenceSynthetic light chain of antibody
52M51 4Met Ala Trp Ile Ser Leu Ile Leu Ser Leu Leu Ala Leu Ser Ser
Gly1 5 10 15Ala Ile Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr
Thr Ser 20 25 30Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr
Gly Ala Val 35 40 45Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys
Pro Asp His Leu 50 55 60Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg
Ala Pro Gly Val Pro65 70 75 80Ala Arg Phe Ser Gly Ser Leu Ile Gly
Asp Lys Ala Ala Leu Thr Ile 85 90 95Thr Gly Ala Gln Thr Glu Asp Glu
Ala Ile Tyr Phe Cys Ala Leu Trp 100 105 110Tyr Ser Asn His Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 115 120 125Gly Gln Pro Lys
Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 130 135 140Glu Glu
Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp145 150 155
160Phe Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro
165 170 175Val Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser
Asn Asn 180 185 190Lys Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala
Arg Ala Trp Glu 195 200 205Arg His Ser Ser Tyr Ser Cys Gln Val Thr
His Glu Gly His Thr Val 210 215 220Glu Lys Ser Leu Ser Arg Ala Asp
Cys Ser225 2305387DNAArtificial SequenceSynthetic light chain of
antibody 52M51 5atggcctgga tttcacttat actctctctc ctggctctca
gctcaggggc catttcccag 60gctgttgtga ctcaggaatc tgcactcacc acatcacctg
gtgaaacagt cacactcact 120tgtcgctcaa gtactggggc tgttacaact
agtaactacg ccaactgggt ccaagaaaaa 180cctgatcatt tattcactgg
tctaataggt ggtaccaaca accgagctcc aggtgttcct 240gccagattct
caggctccct gattggagac aaggctgccc tcaccatcac aggggcacag
300actgaggatg aggcaatata tttctgtgct ctatggtaca gcaaccactg
ggtgttcggt 360ggaggaacca aactgactgt cctaggc 3876180PRTArtificial
SequenceSynthetic light chain of antibody 52M51 6Met Ala Trp Ile
Ser Leu Ile Leu Ser Leu Leu Ala Leu Ser Ser Gly1 5 10 15Ala Ile Ser
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser 20 25 30Pro Gly
Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 35 40 45Thr
Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu 50 55
60Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro65
70 75 80Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile 85 90 95Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala
Leu Trp 100 105 110Tyr Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 115 120 125Gly Gln Pro Lys Ser Ser Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser 130 135 140Glu Glu Leu Glu Thr Asn Lys Ala
Thr Leu Val Cys Thr Ile Thr Asp145 150 155 160Phe Tyr Pro Gly Val
Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro 165 170 175Val Thr Gln
Gly 1807330DNAArtificial SequenceSynthetic light chain of antibody
52M51 7caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac
agtcacactc 60acttgtcgct caagtactgg ggctgttaca actagtaact acgccaactg
ggtccaagaa 120aaacctgatc atttattcac tggtctaata ggtggtacca
acaaccgagc tccaggtgtt 180cctgccagat tctcaggctc cctgattgga
gacaaggctg ccctcaccat cacaggggca 240cagactgagg atgaggcaat
atatttctgt gctctatggt acagcaacca ctgggtgttc 300ggtggaggaa
ccaaactgac tgtcctaggc 3308110PRTArtificial SequenceSynthetic light
chain of antibody 52M51 8Gln Ala Val Val Thr Gln Glu Ser Ala Leu
Thr Thr Ser Pro Gly Glu1 5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser
Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu
Lys Pro Asp His Leu Phe Thr Gly 35 40 45Leu Ile Gly Gly Thr Asn Asn
Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Ile Gly
Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala65 70 75 80Gln Thr Glu Asp
Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95His Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105
11091395DNAArtificial SequenceSynthetic heavy chain of antibody
52M51 9atggaatgga cctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt
ccactcccag 60gttcagctgc agcagtctgg agctgagctg atgaagcctg gggcctcagt
gaagatatcc 120tgcaaggctg ctggctacac aatgagaggc tactggatag
agtggataaa gcagaggcct 180ggacatggcc ttgagtggat tggacagatt
ttacctggaa ctgggagaac taactacaat 240gagaagttca agggcaaggc
cacattcact gcagatacat cctccaacac agccaacatg 300caactcagca
gcctgacatc tgaggactct gccgtctatt actgtgcaag atttgatggt
360aactacggtt actatgctat ggactactgg ggtcaaggat cctcagtcac
cgtctcctca 420gccaaaacga cacccccatc tgtctatcca ctggcccctg
gatctgctgc ccaaactaac 480tccatggtga ccctgggatg cctggtcaag
ggctatttcc ctgagccagt gacagtgacc 540tggaactctg gatccctgtc
cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 600ctctacactc
tgagcagctc agtgactgtc ccctccagcc ctcggcccag cgagaccgtc
660acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat
tgtgcccagg 720gattgtggtt gtaagccttg catatgtaca gtcccagaag
tatcatctgt cttcatcttc 780cccccaaagc ccaaggatgt cctcaccatt
actctgactc ctaaggtcac gtgtgttgtg 840gtagacatca gcaaggatga
tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 900gtgcacacag
ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc
960agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa
atgcagggtc 1020aacagtgcag ctttccctgc ccccatcgag aaaaccatat
ccaaaaccaa aggcagaccg 1080aaggctccac aggtgtacac cattccacct
cccaaggagc agatggccaa ggataaagtc 1140agtctgacct gcatgataac
agacttcttc cctgaagaca taacagtgga gtggcagtgg 1200aatgggcagc
cagcggagaa ctacaagaac actcagccca tcatgaacac gaatggctct
1260tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg
aaatactttc 1320acctgctctg tgttacatga gggcctgcac aaccaccata
ctgagaagag cctctcccac 1380tctcctggta aatga 139510464PRTArtificial
SequenceSynthetic heavy chain of antibody 52M51 10Met Glu Trp Thr
Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly1 5 10 15Val His Ser
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys 20 25 30Pro Gly
Ala Ser Val Lys Ile Ser Cys Lys Ala Ala Gly Tyr Thr Met 35 40 45Arg
Gly Tyr Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu 50 55
60Glu Trp Ile Gly Gln Ile Leu Pro Gly Thr Gly Arg Thr Asn Tyr Asn65
70 75 80Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser
Asn 85 90 95Thr Ala Asn Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val 100 105 110Tyr Tyr Cys Ala Arg Phe Asp Gly Asn Tyr Gly Tyr
Tyr Ala Met Asp 115 120 125Tyr Trp Gly Gln Gly Ser Ser Val Thr Val
Ser Ser Ala Lys Thr Thr 130 135 140Pro Pro Ser Val Tyr Pro Leu Ala
Pro Gly Ser Ala Ala Gln Thr Asn145 150 155 160Ser Met Val Thr Leu
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 165 170 175Val Thr Val
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 180 185 190Phe
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 195 200
205Thr Val Pro Ser Ser Pro Arg Pro Ser Glu Thr Val Thr Cys Asn Val
210 215 220Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val
Pro Arg225 230 235 240Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val
Pro Glu Val Ser Ser 245 250 255Val Phe Ile Phe Pro Pro Lys Pro Lys
Asp Val Leu Thr Ile Thr Leu 260 265 270Thr Pro Lys Val Thr Cys Val
Val Val Asp Ile Ser Lys Asp Asp Pro 275 280 285Glu Val Gln Phe Ser
Trp Phe Val Asp Asp Val Glu Val His Thr Ala 290 295 300Gln Thr Gln
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val305 310 315
320Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe
325 330 335Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu
Lys Thr 340 345 350Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
Val Tyr Thr Ile 355 360 365Pro Pro Pro Lys Glu Gln Met Ala Lys Asp
Lys Val Ser Leu Thr Cys 370 375 380Met Ile Thr Asp Phe Phe Pro Glu
Asp Ile Thr Val Glu Trp Gln Trp385 390 395 400Asn Gly Gln Pro Ala
Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn 405 410 415Thr Asn Gly
Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser 420 425 430Asn
Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly 435 440
445Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
450 455 46011420DNAArtificial SequenceSynthetic heavy chain of
antibody 52M51 11atggaatgga cctgggtctt tctcttcctc ctgtcagtaa
ctgcaggtgt ccactcccag 60gttcagctgc agcagtctgg agctgagctg atgaagcctg
gggcctcagt gaagatatcc 120tgcaaggctg ctggctacac aatgagaggc
tactggatag agtggataaa gcagaggcct 180ggacatggcc ttgagtggat
tggacagatt ttacctggaa ctgggagaac taactacaat 240gagaagttca
agggcaaggc cacattcact gcagatacat cctccaacac agccaacatg
300caactcagca gcctgacatc tgaggactct gccgtctatt actgtgcaag
atttgatggt 360aactacggtt actatgctat ggactactgg ggtcaaggat
cctcagtcac cgtctcctca 42012180PRTArtificial SequenceSynthetic heavy
chain of antibody 52M51 12Met Glu Trp Thr Trp Val Phe Leu Phe Leu
Leu Ser Val Thr Ala Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Met Lys 20 25 30Pro Gly Ala Ser Val Lys Ile Ser
Cys Lys Ala Ala Gly Tyr Thr Met 35 40 45Arg Gly Tyr Trp Ile Glu Trp
Ile Lys Gln Arg Pro Gly His Gly Leu 50 55 60Glu Trp Ile Gly Gln Ile
Leu Pro Gly Thr Gly Arg Thr Asn Tyr Asn65 70 75 80Glu Lys Phe Lys
Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn 85 90 95Thr Ala Asn
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr
Tyr Cys Ala Arg Phe Asp Gly Asn Tyr Gly Tyr Tyr Ala Met Asp 115 120
125Tyr Trp Gly Gln Gly Ser Ser Val Thr Val Ser Ser Ala Lys Thr Thr
130 135 140Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln
Thr Asn145 150 155 160Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly
Tyr Phe Pro Glu Pro 165 170 175Val Thr Val Thr
18013363DNAArtificial SequenceSynthetic heavy chain of antibody
52M51 13caggttcagc tgcagcagtc tggagctgag ctgatgaagc ctggggcctc
agtgaagata 60tcctgcaagg ctgctggcta cacaatgaga ggctactgga tagagtggat
aaagcagagg 120cctggacatg gccttgagtg gattggacag attttacctg
gaactgggag aactaactac 180aatgagaagt tcaagggcaa ggccacattc
actgcagata catcctccaa cacagccaac 240atgcaactca gcagcctgac
atctgaggac tctgccgtct attactgtgc aagatttgat 300ggtaactacg
gttactatgc tatggactac tggggtcaag gatcctcagt caccgtctcc 360tca
36314122PRTArtificial SequenceSynthetic heavy chain of antibody
52M51 14Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly
Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ala Gly Tyr Thr Met Arg
Gly Tyr 20 25 30Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu
Glu Trp Ile 35 40 45Gly Gln Ile Leu Pro Gly Thr Gly Arg Thr Asn Tyr
Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser
Ser Asn Thr Ala Asn65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe Asp Gly Asn Tyr Gly
Tyr Tyr Ala Met Asp Tyr Trp Gly 100
105 110Gln Gly Ser Ser Val Thr Val Ser Ser Ala 115
120156PRTArtificial SequenceSynthetic heavy chain CDR1 antibody
15Arg Gly Tyr Trp Ile Glu1 51617PRTArtificial SequenceSynthetic
heavy chain CDR2 antibody 16Gln Ile Leu Pro Gly Thr Gly Arg Thr Asn
Tyr Asn Glu Lys Phe Lys1 5 10 15Gly1712PRTArtificial
SequenceSynthetic heavy chain CDR3 antibody 17Phe Asp Gly Asn Tyr
Gly Tyr Tyr Ala Met Asp Tyr1 5 101814PRTArtificial
SequenceSynthetic light chain CDR1 antibody 18Arg Ser Ser Thr Gly
Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5 10197PRTArtificial
SequenceSynthetic light chain CDR2 antibody 19Gly Thr Asn Asn Arg
Ala Pro1 52016PRTArtificial SequenceSynthetic light chain CDR3
antibody 20Ala Leu Trp Tyr Ser Asn His Trp Val Phe Gly Gly Gly Thr
Lys Leu1 5 10 15211401DNAArtificial SequenceSynthetic heavy chain
52M51 antibody 21atggattgga catggagggt gttctgcctc ctcgctgtgg
ctcctggagt cctgagccag 60gtccagctcg tccagagcgg ggctgaagtc aagaagcctg
gcgctagcgt caaaatcagc 120tgtaaggtca gcggatacac actgagggga
tactggatcg agtgggtgag gcaggctcca 180ggaaagggcc tggaatggat
cggccagatc ctgcctggaa ccggaaggac aaattacaat 240gagaagttta
agggaagggt cacaatgaca gcagacacaa gcacagacac agcttatatg
300gaactcagct ccctcagatc cgaggacacc gctgtctact attgtgccag
gttcgatgga 360aattacggat actatgccat ggattactgg ggacagggga
caacggtcac cgtgagctca 420gccagcacaa agggccctag cgtcttccct
ctggctccct gcagcaggag caccagcgag 480agcacagccg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540tggaactcag
gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca
600ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg
cacccagacc 660tacacctgca acgtagatca caagcccagc aacaccaagg
tggacaagac agttgagcgc 720aaatgttgtg tcgagtgccc accgtgccca
gcaccacctg tggcaggacc gtcagtcttc 780ctcttccccc caaaacccaa
ggacaccctc atgatctccc ggacccctga ggtcacgtgc 840gtggtggtgg
acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc
900gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag
cacgttccgt 960gtggtcagcg tcctcaccgt tgtgcaccag gactggctga
acggcaagga gtacaagtgc 1020aaggtctcca acaaaggcct cccagccccc
atcgagaaaa ccatctccaa aaccaaaggg 1080cagccccgag aaccacaggt
gtacaccctg cccccatccc gggaggagat gaccaagaac 1140caggtcagcc
tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg
1200gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct
ggactccgac 1260ggctccttct tcctctacag caagctcacc gtggacaaga
gcaggtggca gcaggggaac 1320gtcttctcat gctccgtgat gcatgaggct
ctgcacaacc actacacgca gaagagcctc 1380tccctgtctc cgggtaaatg a
140122466PRTArtificial SequenceSynthetic heavy chain 52M51 antibody
22Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly1
5 10 15Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr
Thr Leu 35 40 45Arg Gly Tyr Trp Ile Glu Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 50 55 60Glu Trp Ile Gly Gln Ile Leu Pro Gly Thr Gly Arg
Thr Asn Tyr Asn65 70 75 80Glu Lys Phe Lys Gly Arg Val Thr Met Thr
Ala Asp Thr Ser Thr Asp 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Phe Asp
Gly Asn Tyr Gly Tyr Tyr Ala Met Asp 115 120 125Tyr Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu145 150 155
160Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
165 170 175Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr 180 185 190Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val 195 200 205Val Thr Val Pro Ser Ser Asn Phe Gly Thr
Gln Thr Tyr Thr Cys Asn 210 215 220Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Thr Val Glu Arg225 230 235 240Lys Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 245 250 255Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280
285Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
290 295 300Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Phe Arg305 310 315 320Val Val Ser Val Leu Thr Val Val His Gln Asp
Trp Leu Asn Gly Lys 325 330 335Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ala Pro Ile Glu 340 345 350Lys Thr Ile Ser Lys Thr Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp385 390 395
400Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met
405 410 415Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp 420 425 430Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His 435 440 445Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro 450 455 460Gly Lys46523141PRTArtificial
SequenceSynthetic heavy chain 52M51 antibody 23Met Asp Trp Thr Trp
Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly1 5 10 15Val Leu Ser Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala
Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Leu 35 40 45Arg Gly
Tyr Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu
Trp Ile Gly Gln Ile Leu Pro Gly Thr Gly Arg Thr Asn Tyr Asn65 70 75
80Glu Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Asp
85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val 100 105 110Tyr Tyr Cys Ala Arg Phe Asp Gly Asn Tyr Gly Tyr Tyr
Ala Met Asp 115 120 125Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Ala 130 135 14024122PRTArtificial SequenceSynthetic heavy chain
52M51 antibody 24Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr
Thr Leu Arg Gly Tyr 20 25 30Trp Ile Glu Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45Gly Gln Ile Leu Pro Gly Thr Gly Arg
Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Ala
Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe Asp Gly
Asn Tyr Gly Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser Ala 115 12025720DNAArtificial
SequenceSynthetic light chain 52M51 antibody 25atgagcgtcc
ctacaatggc ttggatgatg ctcctgctgg gactcctggc ttatggaagc 60ggagtggata
gccaggccgt cgtcacacag gaacctagcc tcaccgttag ccctggagga
120acagtcacac tgacctgtag gagctccaca ggagctgtga caacaagcaa
ttacgctaac 180tggttccagc agaagcccgg tcaagcccct agaaccctca
tcggcggcac caataacaga 240gctcccggag tccccgccag gttctccggc
tccctcctgg gtggcaaggc tgctctgaca 300ctcagcggtg cccagccaga
ggatgaagcg gagtactact gtgcactgtg gtacagcaac 360cattgggttt
tcggaggcgg aacaaagtta accgtcctcg ggcagcctaa ggctgctcct
420agcgtcacac tgttcccccc atctagcgag gagctgcagg ctaacaaggc
aaccctcgtc 480tgcctggtta gcgacttcta ccctggcgct gtcacagtgg
cctggaaagc tgacggctcc 540cctgtgaaag ttggcgtcga aaccacaaag
ccttctaagc agagcaataa taaatatgcc 600gcaagctcct acctctccct
gactcctgag cagtggaaaa gccataggag ctactcctgc 660cgggtcacac
acgaaggaag cacagtggaa aagacagtcg cccctgctga gtgtagctga
72026239PRTArtificial SequenceSynthetic light chain 52M51 antibody
26Met Ser Val Pro Thr Met Ala Trp Met Met Leu Leu Leu Gly Leu Leu1
5 10 15Ala Tyr Gly Ser Gly Val Asp Ser Gln Ala Val Val Thr Gln Glu
Pro 20 25 30Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys
Arg Ser 35 40 45Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp
Phe Gln Gln 50 55 60Lys Pro Gly Gln Ala Pro Arg Thr Leu Ile Gly Gly
Thr Asn Asn Arg65 70 75 80Ala Pro Gly Val Pro Ala Arg Phe Ser Gly
Ser Leu Leu Gly Gly Lys 85 90 95Ala Ala Leu Thr Leu Ser Gly Ala Gln
Pro Glu Asp Glu Ala Glu Tyr 100 105 110Tyr Cys Ala Leu Trp Tyr Ser
Asn His Trp Val Phe Gly Gly Gly Thr 115 120 125Lys Leu Thr Val Leu
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu 130 135 140Phe Pro Pro
Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val145 150 155
160Cys Leu Val Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys
165 170 175Ala Asp Gly Ser Pro Val Lys Val Gly Val Glu Thr Thr Lys
Pro Ser 180 185 190Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr 195 200 205Pro Glu Gln Trp Lys Ser His Arg Ser Tyr
Ser Cys Arg Val Thr His 210 215 220Glu Gly Ser Thr Val Glu Lys Thr
Val Ala Pro Ala Glu Cys Ser225 230 23527134PRTArtificial
SequenceSynthetic light chain 52M51 antibody 27Met Ser Val Pro Thr
Met Ala Trp Met Met Leu Leu Leu Gly Leu Leu1 5 10 15Ala Tyr Gly Ser
Gly Val Asp Ser Gln Ala Val Val Thr Gln Glu Pro 20 25 30Ser Leu Thr
Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser 35 40 45Ser Thr
Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Phe Gln Gln 50 55 60Lys
Pro Gly Gln Ala Pro Arg Thr Leu Ile Gly Gly Thr Asn Asn Arg65 70 75
80Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys
85 90 95Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu
Tyr 100 105 110Tyr Cys Ala Leu Trp Tyr Ser Asn His Trp Val Phe Gly
Gly Gly Thr 115 120 125Lys Leu Thr Val Leu Gly
13028115PRTArtificial SequenceSynthetic light chain 52M51 antibody
28Ser Gly Val Asp Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr1
5 10 15Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr
Gly 20 25 30Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Phe Gln Gln Lys
Pro Gly 35 40 45Gln Ala Pro Arg Thr Leu Ile Gly Gly Thr Asn Asn Arg
Ala Pro Gly 50 55 60Val Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly
Lys Ala Ala Leu65 70 75 80Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu
Ala Glu Tyr Tyr Cys Ala 85 90 95Leu Trp Tyr Ser Asn His Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr 100 105 110Val Leu Gly
11529720DNAArtificial SequenceSynthetic light chain 52M51 antibody
29atgagcgtcc ctacaatggc ttggatgatg ctcctgctgg gactcctggc ttatggaagc
60ggagtggata gccagaccgt cgtcacacag gaacctagct tttccgttag ccctggagga
120acagtcacac tgacctgtag gagctccaca ggagctgtga caacaagcaa
ttacgctaac 180tggtatcagc agactcccgg tcaagcccct agaaccctca
tcggcggcac caataacaga 240gctcccggag tccccgacag gttctccggc
tccatcctgg gaaataaagc tgctctgaca 300atcacaggtg cccaggctga
cgatgaaagc gactactact gtgcactgtg gtacagcaac 360cattgggttt
tcggaggcgg aacaaagtta accgtcctcg ggcagcctaa ggctgctcct
420agcgtcacac tgttcccccc atctagcgag gagctgcagg ctaacaaggc
aaccctcgtc 480tgcctggtta gcgacttcta ccctggcgct gtcacagtgg
cctggaaagc tgacggctcc 540cctgtgaaag ttggcgtcga aaccacaaag
ccttctaagc agagcaataa taaatatgcc 600gcaagctcct acctctccct
gactcctgag cagtggaaaa gccataggag ctactcctgc 660cgggtcacac
acgaaggaag cacagtggaa aagacagtcg cccctgctga gtgtagctga
72030239PRTArtificial SequenceSynthetic light chain 52M51 antibody
30Met Ser Val Pro Thr Met Ala Trp Met Met Leu Leu Leu Gly Leu Leu1
5 10 15Ala Tyr Gly Ser Gly Val Asp Ser Gln Thr Val Val Thr Gln Glu
Pro 20 25 30Ser Phe Ser Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys
Arg Ser 35 40 45Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp
Tyr Gln Gln 50 55 60Thr Pro Gly Gln Ala Pro Arg Thr Leu Ile Gly Gly
Thr Asn Asn Arg65 70 75 80Ala Pro Gly Val Pro Asp Arg Phe Ser Gly
Ser Ile Leu Gly Asn Lys 85 90 95Ala Ala Leu Thr Ile Thr Gly Ala Gln
Ala Asp Asp Glu Ser Asp Tyr 100 105 110Tyr Cys Ala Leu Trp Tyr Ser
Asn His Trp Val Phe Gly Gly Gly Thr 115 120 125Lys Leu Thr Val Leu
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu 130 135 140Phe Pro Pro
Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val145 150 155
160Cys Leu Val Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys
165 170 175Ala Asp Gly Ser Pro Val Lys Val Gly Val Glu Thr Thr Lys
Pro Ser 180 185 190Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr 195 200 205Pro Glu Gln Trp Lys Ser His Arg Ser Tyr
Ser Cys Arg Val Thr His 210 215 220Glu Gly Ser Thr Val Glu Lys Thr
Val Ala Pro Ala Glu Cys Ser225 230 23531134PRTArtificial
SequenceSynthetic light chain 52M51 antibody 31Met Ser Val Pro Thr
Met Ala Trp Met Met Leu Leu Leu Gly Leu Leu1 5 10 15Ala Tyr Gly Ser
Gly Val Asp Ser Gln Thr Val Val Thr Gln Glu Pro 20 25 30Ser Phe Ser
Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser 35 40 45Ser Thr
Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Tyr Gln Gln 50 55 60Thr
Pro Gly Gln Ala Pro Arg Thr Leu Ile Gly Gly Thr Asn Asn Arg65 70 75
80Ala Pro Gly Val Pro Asp Arg Phe Ser Gly Ser Ile Leu Gly Asn Lys
85 90 95Ala Ala Leu Thr Ile Thr Gly Ala Gln Ala Asp Asp Glu Ser Asp
Tyr 100 105 110Tyr Cys Ala Leu Trp Tyr Ser Asn His Trp Val Phe Gly
Gly Gly Thr 115 120 125Lys Leu Thr Val Leu Gly
13032115PRTArtificial SequenceSynthetic light chain 52M51 antibody
32Ser Gly Val Asp Ser Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser1
5 10 15Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr
Gly 20 25 30Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Tyr Gln Gln Thr
Pro Gly 35 40 45Gln Ala Pro Arg Thr Leu Ile Gly Gly Thr Asn Asn Arg
Ala Pro Gly 50 55 60Val Pro Asp Arg Phe Ser Gly Ser Ile Leu Gly Asn
Lys Ala Ala Leu65 70 75 80Thr Ile Thr Gly Ala Gln Ala Asp Asp Glu
Ser Asp Tyr Tyr Cys Ala 85 90 95Leu Trp Tyr Ser Asn His Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr 100 105 110Val Leu Gly 115
* * * * *
References